Methods of use for antibodies against parathyroid hormone

ABSTRACT

Embodiments of the invention described herein relate to antibodies directed to the antigen parathyroid hormone (PTH) and uses of such antibodies. In particular, in some embodiments, there are provided fully human monoclonal antibodies directed to the antigen PTH. In further embodiments, nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDRs), specifically from FR1 through FR4 or CDR1 through CDR3, are provided.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 10/638,265 titled ANTIBODIES DIRECTED TOPARATHYROID HORMONE (PTH) AND USES THEREOF filed Aug. 8, 2003 now U.S.Pat. No. 7,288,253.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention described herein relate to antibodiesdirected to the antigen parathyroid hormone (PTH), particular epitopesof PTH, and uses of such antibodies. In particular, in accordance withembodiments of the invention described herein, there are provided fullyhuman monoclonal antibodies (mAbs) directed to the antigen PTH.Nucleotide sequences encoding, and amino acid sequences comprising,heavy and light chain immunoglobulin molecules, particularly sequencescorresponding to contiguous heavy and light chain sequences spanning theframework regions and/or complementarity determining regions (CDRs),specifically from FR1 through FR4 or CDR1 through CDR3, are provided.The antibodies of the invention find use as diagnostics and astreatments for diseases associated with the overproduction of PTH.

2. Description of the Related Art

Parathyroid glands are part of the endocrine system and produceparathyroid hormone (PTH). PTH regulates the levels of calcium,phosphorus, and magnesium, in the bloodstream, maintaining anappropriate balance of these substances, which is essential for normalbone mineralization. Chronic, excessive production of PTH is known ashyperparathyroidism (HPT). Overproduction of parathyroid hormone leadsto an elevated blood calcium level and decreased blood phosphate level.Calcium is removed from bones and calcium absorption from thegastrointestinal (GI) tract increases. The kidneys attempt to compensatefor the increased blood calcium level by secreting excess calcium in theurine, which can result in the formation of kidney stones. The effectsof increased PTH levels are seen not only in the kidneys, but also inthe skeleton, stomach and intestines, the nervous system, and themuscles (R. S. Cotran et al., eds., Robbins Pathologic Basis of Disease1246-47 (4th ed., W.B. Saunders Co., Philadelphia 1989).

In primary hyperparathyroidism, the increased secretion of PTH occursbecause of the presence of a tumor, a parathyroid adenoma (˜80%), orless commonly by hyperplasia of the parathyroid (˜15%) or carcinoma(˜5%). As a result of elevated blood calcium levels, symptoms caninclude kidney stones, bone pain, fatigue, anorexia, nausea and vomiting(L. M. Tierney, Jr., et al., eds., Current Medical Diagnosis andTreatment 1001-02 (35th ed., Appleton & Lange, Stamford, Conn. 1996)).Current medical management of primary HPT is not satisfactory becausepresently, there are no agents that can produce sustained blockage ofPTH release by the parathyroid glands. Surgical removal of part or allof the parathyroid glands is the preferred treatment, althoughcomplications such as damage to the laryngeal nerve and prolongedhypocalcemia can occur postoperatively.

In secondary hyperparathyroidism, the excess production of PTH isnormally a result of either vitamin D deficiencies (rickets andosteomalacia) or chronic renal failure (CRF). When secondary HPT is dueto renal failure, the pathology is characterized by hypocalcemia andhyperphosphatemia and a relative inability to respond to PTH. Thisresistance to PTH function leads to hyperplasia of the parathyroidglands and excessive production of PTH as the glands try to re-establishnormocalcemia and normophosphatemia. The resistance to PTH levels is dueto a failure to produce calcitriol (active form of vitamin D) in thekidneys and a failure to excrete phosphate through the kidneys.Calcitriol acts directly on parathyroid glands to inhibit PTH productionand the GI tract to promote calcium absorption. Therefore the loss ofcalcitriol leads to increased serum PTH levels. High phosphate levelsalso act directly on parathyroid tissue to induce the expression of PTHand can interact directly with calcium to maintain hypocalcemia. Theloss of these negative feedback mechanisms account for most of theresistance to PTH seen in CRF (Fauci, A. S. et al., eds., Harrison'sPrinciples of Internal Medicine 2214-47 (14th ed., McGraw-Hill Co.1998)). In severe secondary HPT, extremely high levels of PTH overwhelmthe bone's resistance to the hormone resulting in high serum calcium andphosphate levels that may cause diffuse calcification in the skin, softtissues, and arteries (calciphylaxis). Such calcification can result inpainful ischemic necrosis of the skin and gangrene, cardiac arrhythmias,and pulmonary failure (Tierney et al., supra at 1003).

Currently, secondary HPT is treated medically with phosphate binderssuch as calcium carbonate and with supraphysiological levels of vitaminD analogues such as calcitriol and doxercalciferol. Not all patientsrespond to calcitriol and hypercalcemia is a common complication oftreatment (Felsenfeld, A. J., J. Am. Soc. Nephrology 8(6):993-1004(1997)). Calcimimetics, designed as allosteric modulators of the calciumreceptor, are also in clinical development as a possible therapy.

PTH is an 84-amino-acid peptide secreted from the parathyroid glands.Its amino acid sequence (Keutman, H. T. et al., Biochemistry 17:5723-29(1978)) and the nucleotide sequence of the related gene (Hendy et al.,Proc. Natl. Acad. Sci. USA 78:7365-69 (1981)) are known. PTH actsthrough the PTH/parathyroid-related protein (PTHrP) receptor to promotebone resorption and decrease calcium excretion. Human parathyroidhormone (hPTH) circulates as substantially intact hPTH1-84 and fragmentsthereof. Full length hPTH1-84 and fragment hPTH1-34 are believed to bebiologically active, while fragment hPTH35-84 is believed to beinactive. Fragments lacking the N-terminus of PTH (hPTH7-84 or hPTH7-34)are not only inactive, but can also inhibit biologically active PTH invivo (Horiuchi et al., Science 220:1053-55 (1983)).

Lindall, in U.S. Pat. No. 4,341,755, describes an antibodyradioimmunoassay of PTH in mammalian serum. A chicken antibody withaffinity for the portion of human PTH corresponding to amino acids 65-84was utilized in the assay. Adermann et al., in U.S. Pat. No. 6,030,790,disclose polyclonal antibodies made by injecting unspecified animalswith various fragments of human PTH. Japan Tobacco, Inc., in JapanesePatent Application No. JP 98337263, filed Nov. 27, 1998, discloses humanmonoclonal antibodies with reactivity to human PTH.

The obstacles to developing a monoclonal or polyclonal antibody to PTHfor therapeutic applications have been described and have beenattributed to inadequate affinity and immunogenicity (Bradwell, A. R. etal., Lancet 353:370-73 (1999)). Bradwell et al. successfully immunized apatient suffering from parathyroid carcinoma with PTH and the patientproduced autoantibodies against PTH. However, due to the clinical needto titrate PTH to an individual target range in the hyperparathyroidpatient population, a clinical immunization approach would not begenerally applicable given the heterogeneity of immune responses and theneed to break tolerance to a self antigen. Thus, the unmet need for atherapeutically useful anti-PTH antibody remains.

SUMMARY OF THE INVENTION

The invention described herein relates to monoclonal antibodies thatbind PTH and affect PTH function. Accordingly, embodiments of theinvention relate to human anti-PTH monoclonal antibodies and anti-PTHmonoclonal antibody preparations with desirable properties fromdiagnostic and therapeutic perspectives. In particular, one embodimentof the invention provides anti-PTH antibodies having characteristicsthat provide therapeutic utility, including, for example, but notlimited to, strong binding affinity for PTH, the ability to neutralizePTH in vitro, and the ability to produce prolonged neutralization of PTHin vivo.

One aspect of the invention is a method of reducing elevated circulatinglevels of parathyroid hormone (PTH) associated with hyperparathyroidism,including: identifying an animal in need of treatment forhyperparathyroidism; and administering to the animal a therapeuticallyeffective dose of a fully human monoclonal antibody that binds with anaffinity to PTH of less than 100 pM, and reduces the circulating levelsof PTH.

Another aspect of the invention is a method of effectively treatinghypercalcemia in a patient, including: identifying a patient in need oftreatment for hypercalcemia; and administering to the patient atherapeutically effective dose of a fully human monoclonal antibody thatspecifically binds to parathyroid hormone (PTH).

Another aspect of the invention is a method of effectively treatingparathyroid carcinoma in a patient, including: identifying a patient inneed of treatment for parathyroid carcinoma; and administering to thepatient a therapeutically effective dose of a fully human monoclonalantibody that specifically binds to parathyroid hormone (PTH).

Another aspect of the invention is an isolated or purified fully humanmonoclonal antibody that binds to an epitope containing amino acids18-34 of parathyroid hormone (PTH).

Another aspect of the invention is an isolated or purified fully humanmonoclonal antibody that binds to PTH with an affinity of less than 100pM.

Another aspect of the invention is a therapeutic composition for thetreatment of hyperparathyroidism, including a fully human monoclonalantibody that binds to an epitope containing amino acids 18-34 ofparathyroid hormone (PTH) in association with a therapeuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing neutralization data for anti-PTH mAb 183 asdetermined by calcium mobilization assay on FLuorometric Imaging PlateReader (FLIPR).

FIG. 2 is a graph showing neutralization of human PTH in vivo byanti-PTH mAb 183 as measured by reversal of a hypercalcemic response toinfused human PTH (34 mer) in rats.

FIG. 3 is an amino acid sequence alignment of heavy chain variableregions of anti-PTH mAbs generated according to the invention with theirassociated germline variable region sequences.

FIG. 4 is an amino acid alignment of light chain variable regions ofanti-PTH mAbs generated according to the invention with their associatedgermline variable region sequences.

FIG. 5A is a line graph reporting the results of an experiment whereinmonkeys were treated with an anti-PTH antibody or a control, wherein thetotal calcium levels in the animal over time were measured.

FIG. 5B is a line graph reporting the results of an experiment whereinmonkeys were treated with an anti-PTH antibody or a control, wherein theionized calcium levels in the animal over time were measured.

FIG. 6 is a line graph reporting the results of an experiment whereinmonkeys were treated with an anti-PTH antibody or a control, wherein thefree serum PTH levels were measured over time.

FIG. 7 is a line graph reporting the results of an experiment whereinmonkeys were treated with anti-PTH antibody 183 alone or antibody 183 incombination with antibody sc275, wherein the percent change of serum iCais measured over time.

FIG. 8 is a line graph reporting the results of an experiment in which apatient with parathyroid carcinoma was treated with mAb 183, whereinserum calcium and unbound iPTH are measured over time.

FIG. 9 is a line graph reporting the results of an experiment in which apatient with parathyroid carcinoma was treated with mAb 183, whereinserum calcium and unbound iPTH are measured over time.

FIG. 10 is a line graph reporting the results of an experiment in whicha patient with parathyroid carcinoma was treated with mAb 183, wherein1, 25 dihydroxy vitamin D and unbound iPTH are measured over time.

FIG. 11 is a line graph reporting the results of an experiment in whicha patient with parathyroid carcinoma was treated with mAb 183, whereinplasma N-telopeptides and unbound iPTH are measured over time.

FIG. 12 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein unbound iPTH is measured over time.

FIG. 13 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein ionized calcium is measured over time.

FIG. 14 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein serum cCa is measured over time.

FIG. 15 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein percent change of serum cCa is measured over time.

FIG. 16 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein unbound iPTH is measured over time.

FIG. 17 is a bar graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein the percentage of patients exhibiting a particular level ofunbound iPTH is reported based on the dosage administered.

FIG. 18 is a bar graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein the mean change in bAP is reported based on the dosageadministered.

FIG. 19 is a line graph reporting the results of an experiment in whichpatients with Secondary Hyperparathyroidism (SHPT) were treated with mAb183, wherein mean serum mAb 183 concentration is measured over time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Embodiments of the invention described herein relate to fully humananti-PTH antibodies and their uses. Such fully human antibodies have theadvantage of improved pharmacokinetic and safety profiles relative toantibodies containing non-human sequences and, accordingly,immunogenicity in humans is not anticipated. Through use of adual-antigen immunization strategy combined with the screeningtechnology described herein, monoclonal antibodies with rare affinityand prolonged duration of action in vivo have been discovered that haveutility in therapeutic applications. Additionally, the in vivoneutralization studies provided herein demonstrate that for an anti-PTHantibody to be highly therapeutically viable, it should possess highaffinity, preferably in the nanomolar range and more preferably in thepicomolar range. The anti-PTH antibodies of the invention describedherein have been found to preferentially and specifically bind to PTH.

One embodiment of the invention relates to therapeutic antibodies thatbind to the epitope comprising amino acids 18-34 of PTH whichcorresponds to the amino acid sequence “MERVEWLRKKLQDVHNF” (SEQ ID NO:98) As discussed below, it was discovered that antibodies, such asantibody 183, that bind to this epitope have therapeutic value fortreating PTH-related diseases in humans.

Another embodiment of the invention relates to the use of therapeuticantibodies that bind to the epitope “RVEWLRKKL” (SEQ ID NO: 99), whichcorresponds to amino acids 20-28 of PTH. Yet another embodiment of theinvention relates to antibodies that bind to a subspecies of thisepitope, wherein therapeutically effective antibodies bind to an epitopehaving the amino acid sequence “RVEXLRXKL” (SEQ ID NO: 100)corresponding to amino acids 20, 21, 22, 24, 25, 27 and 28 of PTH, andwherein “X” can be any amino acid. In one embodiment, “X” is the aminoacid alanine.

Other embodiments of the invention relate to therapeutically effectiveantibodies that bind to amino acids “SVSEIQL” (SEQ ID NO: 101)corresponding to an epitope at amino acids 1-7 of PTH. In oneembodiment, the antibody is the sc275 antibody discussed below.

Still another embodiment of the invention relates to the discovery of asynergistic effect in lowering Ca levels in a mammal by the combinedtreatment of an antibody that binds to the epitope RVEXLRXKL (SEQ ID NO:100) in combination with a secondary antibody that binds to a differentepitope of PTH. For example, it was discovered that such a combinationtreatment resulted in a significantly longer duration of reduced serumcalcium levels in vivo. In addition, the extent to which serum calciumwas reduced was far greater in animals given the combination treatmentin comparison to animals that were given one antibody alone. In oneembodiment, the second antibody binds to the epitope SVSEIQL (SEQ ID NO:101) of PTH. However, embodiments of the invention are not limited tothe second antibody binding to any particular epitope in order todiscover the synergistic effect. In one embodiment, the combinationtreatment results in at least 10 days of reduced serum calcium levels.In another embodiment, the combination treatment results in at least 15days of reduced serum calcium levels. In still another embodiment, thecombination treatment results in at least 20, and more preferably, 25and most preferably, 30 days of reduced serum calcium levels in amammal.

Accordingly, embodiments of the invention provide isolated antibodies,or fragments of those antibodies, that bind to PTH. As known in the art,the antibodies can advantageously be, e.g., monoclonal, chimeric and/orhuman antibodies. Embodiments of the invention also provide cells forproducing these antibodies.

In addition, embodiments of the invention provide for using theseantibodies as a diagnostic or treatment for disease. For example,embodiments of the invention provide methods and antibodies forinhibition expression of PTH associated with hyperparathyroidism.Preferably, the antibodies are used to treat primary and secondaryhyperparathyroidism. In association with such treatment, articles ofmanufacture comprising antibodies of the invention described herein areprovided. Additionally, an assay kit comprising antibodies in accordancewith the invention described herein is provided to screen forhyperparathyroidism.

Antibodies of the invention described herein, such as anti-PTH mAb 183antibody, possess high affinity, significant neutralization potential,and sustained half-life and prolonged duration of action. Anti-PTHantibodies in accordance with the invention described herein, such asanti-PTH mAb 183 antibody, reduce the level of unbound PTH in normalSprague-Dawley rats receiving 50 μg/kg/day human PTH (1-34) bysubcutaneous Alzet osmotic pump by at least 50% for at least 48 hoursfollowing a single 3 mg/kg intravenous dose of antibody, as measured bydirect assay or by a biomarker of PTH bioactivity.

Additionally, as reported below, it was discovered that cynomolgusmonkeys infused with intact cynomolgus parathyroid hormone (cynoPTH(1-84)). to mimic the effects of hypercalcemia were found to have asustained, dose-dependent suppression of hypercalcemia followingtreatment with anti-PTH mAb 183. The study confirms that anti-PTH mAb183 neutralizes the bioactivity of intact PTH in primates, and confirmedthe results found in rats infused with the human PTH (1-34) N-terminalfragment.

In another study, discussed in detail below, a patient withhyperparathyroidism secondary to parathyroid carcinoma was treated withmAb 183. The patient continued to receive 3-4 times weekly hemodialysisand intermittent doses of zoledronic acid. Treatment with mAb 183resulted in a profound, dose- and exposure-dependent reduction in plasmaunbound PTH from a pre-dose level of 1196 pg/ml to the normal range(range 30 pg/ml-100 pg/ml) at a final dose of 200 mg per week. PlasmaNTx levels were reduced following each dose of mAb 183 suggesting boneturnover was impacted by the treatment. In addition, vitamin D levelswere reduced by the treatment. Serum iCa fluctuated with hemodialysisbut remained elevated. No drug related toxicities were observed duringor after the administration of mAb 183 and the patient reportedimprovement of his bone pain. These findings support the potentialbenefit of the use of antibodies against PTH in the treatment ofhyperparathyroidism secondary to parathyroid carcinoma.

Accordingly, antibodies of the invention described herein possesstherapeutic utilities. For example, a single dose of antibodies inaccordance with the invention, such as anti-PTH mAb 183 antibody, willproduce a reduction of unbound PTH levels in serum of a patient fromhyperparathyroid levels to a normal or near-normal level for 24 to 36hours, preferably 48 to 60 hours, and more preferably 72 to 84 hours.Similarly, administration of at least one dose of an anti-PTH antibodyof the invention, such as anti-PTH mAb 183 antibody, is capable ofreducing circulating PTH levels in a patient by about 25%, preferably byabout 50%, and more preferably by about 75% relative to the levels priorto administration and preferably maintain such reduction for a period ofabout 24 to 36 hours, preferably for a period of about 48 to 60 hours,and more preferably for a period of about 72 to 84 hours.

Further embodiments, features, and the like regarding the antibodies ofthe invention are provided in additional detail below.

Sequence Listing

The heavy chain and light chain variable region nucleotide and aminoacid sequences of representative human anti-PTH antibodies are providedin the sequence listing, the contents of which are summarized in Table 1below.

TABLE 1 mAb SEQ ID ID No.: Sequence NO: 183 Nucleotide sequence encodingthe variable region of the heavy chain 1 Amino acid sequence encodingthe variable region of the heavy chain 2 Nucleotide sequence encodingthe variable region of the light chain 3 Amino acid sequence encodingthe variable region of the light chain 4 262 Nucleotide sequenceencoding the variable region of the heavy chain 5 Amino acid sequenceencoding the variable region of the heavy chain 6 Nucleotide sequenceencoding the variable region of the light chain 7 Amino acid sequenceencoding the variable region of the light chain 8  57 Nucleotidesequence encoding the variable region of the heavy chain 9 Amino acidsequence encoding the variable region of the heavy chain 10 Nucleotidesequence encoding the variable region of the light chain 11 Amino acidsequence encoding the variable region of the light chain 12  45Nucleotide sequence encoding the variable region of the heavy chain 13Amino acid sequence encoding the variable region of the heavy chain 14Nucleotide sequence encoding the variable region of the light chain 15Amino acid sequence encoding the variable region of the light chain 16026 Nucleotide sequence encoding the variable region of the heavy chain17 Amino acid sequence encoding the variable region of the heavy chain18 Nucleotide sequence encoding the variable region of the light chain19 Amino acid sequence encoding the variable region of the light chain20 140 Nucleotide sequence encoding the variable region of the heavychain 21 Amino acid sequence encoding the variable region of the heavychain 22 Nucleotide sequence encoding the variable region of the lightchain 23 Amino acid sequence encoding the variable region of the lightchain 24  11 Nucleotide sequence encoding the variable region of theheavy chain 25 Amino acid sequence encoding the variable region of theheavy chain 26 Nucleotide sequence encoding the variable region of thelight chain 27 Amino acid sequence encoding the variable region of thelight chain 28  86 Nucleotide sequence encoding the variable region ofthe heavy chain 29 Amino acid sequence encoding the variable region ofthe heavy chain 30 Nucleotide sequence encoding the variable region ofthe light chain 31 Amino acid sequence encoding the variable region ofthe light chain 32 124 Nucleotide sequence encoding the variable regionof the heavy chain 33 Amino acid sequence encoding the variable regionof the heavy chain 34 Nucleotide sequence encoding the variable regionof the light chain 35 Amino acid sequence encoding the variable regionof the light chain 36 275 Nucleotide sequence encoding the variableregion of the heavy chain 37 Amino acid sequence encoding the variableregion of the heavy chain 38 Nucleotide sequence encoding the variableregion of the light chain 39 Amino acid sequence encoding the variableregion of the light chain 40  96 Nucleotide sequence encoding thevariable region of the heavy chain 41 Amino acid sequence encoding thevariable region of the heavy chain 42 Nucleotide sequence encoding thevariable region of the light chain 43 Amino acid sequence encoding thevariable region of the light chain 44 238 Nucleotide sequence encodingthe variable region of the heavy chain 45 Amino acid sequence encodingthe variable region of the heavy chain 46 Nucleotide sequence encodingthe variable region of the light chain 47 Amino acid sequence encodingthe variable region of the light chain 48 214 Nucleotide sequenceencoding the variable region of the heavy chain 49 Amino acid sequenceencoding the variable region of the heavy chain 50 Nucleotide sequenceencoding the variable region of the light chain 51 Amino acid sequenceencoding the variable region of the light chain 52 225 Nucleotidesequence encoding the variable region of the heavy chain 53 Amino acidsequence encoding the variable region of the heavy chain 54 Nucleotidesequence encoding the variable region of the light chain 55 Amino acidsequence encoding the variable region of the light chain 56 195Nucleotide sequence encoding the variable region of the heavy chain 57Amino acid sequence encoding the variable region of the heavy chain 58Nucleotide sequence encoding the variable region of the light chain 59Amino acid sequence encoding the variable region of the light chain 60168 Nucleotide sequence encoding the variable region of the heavy chain61 Amino acid sequence encoding the variable region of the heavy chain62 Nucleotide sequence encoding the variable region of the light chain63 Amino acid sequence encoding the variable region of the light chain64 163 Nucleotide sequence encoding the variable region of the heavychain 65 Amino acid sequence encoding the variable region of the heavychain 66 Nucleotide sequence encoding the variable region of the lightchain 67 Amino acid sequence encoding the variable region of the lightchain 68 113 Nucleotide sequence encoding the variable region of theheavy chain 69 Amino acid sequence encoding the variable region of theheavy chain 70 Nucleotide sequence encoding the variable region of thelight chain 71 Amino acid sequence encoding the variable region of thelight chain 72 302 Nucleotide sequence encoding the variable region ofthe heavy chain 73 Amino acid sequence encoding the variable region ofthe heavy chain 74 Nucleotide sequence encoding the variable region ofthe light chain 75 Amino acid sequence encoding the variable region ofthe light chain 76 168g2/ Nucleotide sequence encoding the variableregion of the heavy chain 77 183k Amino acid sequence encoding thevariable region of the heavy chain 78 Nucleotide sequence encoding thevariable region of the light chain 79 Amino acid sequence encoding thevariable region of the light chain 80Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the invention described herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1989), which is incorporatedherein by reference. The nomenclatures utilized in connection with, andthe laboratory procedures and techniques of, analytical chemistry,synthetic organic chemistry, and medicinal and pharmaceutical chemistrydescribed herein are those well known and commonly used in the art.Standard techniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g. free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa light chainimmunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. See M.O. Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, 101-110and Supplement 2 to Vol. 5, 1-10 (National Biomedical ResearchFoundation 1972). The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. The term“corresponds to” is used herein to mean that a polynucleotide sequenceis homologous (i.e., is identical, not strictly evolutionarily related)to all or a portion of a reference polynucleotide sequence, or that apolypeptide sequence is identical to a reference polypeptide sequence.In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence,” “comparison window,” “sequence identity,” “percentage ofsequence identity,” and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window,” as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ded., Golub, E. S. and Gren, D. R. eds., Sinauer Associates, Sunderland,Mass. 1991), which is incorporated herein by reference. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as α-, α-disubstituted amino acids, N-alkyl aminoacids, lactic acid, and other unconventional amino acids may also besuitable components for polypeptides of the invention described herein.Examples of unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline). In the polypeptide notation usedherein, the left-hand direction is the amino terminal direction and theright-hand direction is the carboxy-terminal direction, in accordancewith standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions that are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the invention described herein, providing that thevariations in the amino acid sequence maintain at least 75%, morepreferably at least 80%, 90%, 95%, and most preferably 99%. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. More preferred families are:serine and threonine are aliphatic-hydroxy family; asparagine andglutamine are an amide-containing family; alanine, valine, leucine andisoleucine are an aliphatic family; and phenylalanine, tryptophan, andtyrosine are an aromatic family. For example, it is reasonable to expectthat an isolated replacement of a leucine with an isoleucine or valine,an aspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Assays are described in detailherein. Fragments or analogs of antibodies or immunoglobulin moleculescan be readily prepared by those of ordinary skill in the art. Preferredamino- and carboxy-termini of fragments or analogs occur near boundariesof functional domains. Structural and functional domains can beidentified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal., Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, ed., W.H. Freeman andCompany, New York 1984); Introduction to Protein Structure (Branden, C.and Tooze, J. eds., Garland Publishing, New York, N.Y. 1991); andThornton et al., Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to a PTH, under suitablebinding conditions, (2) ability to block appropriate PTH binding, or (3)ability to inhibit PTH expressing cell growth in vitro or in vivo.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to the naturallyoccurring sequence. Analogs typically are at least 20 amino acids long,preferably at least 50 amino acids long or longer, and can often be aslong as a full-length naturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger, TINS p. 392 (1985); and Evans et al., J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the dissociation constant is ≦1 μM, preferably ≦100 nM and mostpreferably ≦10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Active” or “activity” for the purposes herein refers to form(s) of PTHpolypeptide which retain a biological and/or an immunological activityof native or naturally occurring PTH polypeptides, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally occurring PTH polypeptideother than the ability to induce the production of an antibody againstan antigenic epitope possessed by a native or naturally occurring PTHpolypeptide and an “immunological” activity refers to the ability toinduce the production of an antibody against an antigenic epitopepossessed by a native or naturally occurring PTH polypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Mammal” refers to any animal classified as a mammal, including humans,other primates, such as monkeys, chimpanzees and gorillas, domestic andfarm animals, and zoo, sports, laboratory, or pet animals, such as dogs,cats, cattle, horses, sheep, pigs, goats, rabbits, rodents, etc. Forpurposes of treatment, the mammal is preferably human.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an “F(ab′)₂” fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and binding site of the antibody. This regionconsists of a dimer of one heavy- and one light-chain variable domain intight, non-covalent association. It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the VH-VL dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, forexample, even a single variable domain (e.g., the VH or VL portion ofthe Fv dimer or half of an Fv comprising only three CDRs specific for anantigen) may have the ability to recognize and bind antigen, although,possibly, at a lower affinity than the entire binding site.

A Fab fragment also contains the constant domain of the light chain andthe first constant domain (CH1) of the heavy chain. Fab fragments differfrom Fab′ fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. F(ab′)₂ antibody fragments originallywere produced as pairs of Fab′ fragments which have hinge cysteinesbetween them. Other chemical couplings of antibody fragments are alsoknown.

“Solid phase” means a non-aqueous matrix to which the antibodiesdescribed herein can adhere. Examples of solid phases encompassed hereininclude those formed partially or entirely of glass (e.g., controlledpore glass), polysaccharides (e.g., agarose), polyacrylamides,polystyrene, polyvinyl alcohol and silicones. In certain embodiments,depending on the context, the solid phases can comprise the well of anassay plate; in others it is a purification column (e.g., an affinitychromatography column). This term also includes a discontinuous solidphase of discrete particles, such as those described in U.S. Pat. No.4,275,149.

The term “liposome” is used herein to denote a small vesicle composed ofvarious types of lipids, phospholipids and/or surfactant which is usefulfor delivery of a drug (such as a PTH polypeptide or antibody thereto)to a mammal. The components of the liposomes are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

The term “small molecule” is used herein to describe a molecule with amolecular weight below about 500 Daltons.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” includes human and veterinary subjects.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about 50to 70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. Seegenerally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. RavenPress, N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair form theantibody-binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.1991) (1987), or Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987);Chothia et al., Nature 342:878-83 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315-21 (1990);Kostelny et al., J. Immunol. 148:1547-53 (1992). Production ofbispecific antibodies can be a relatively labor intensive processcompared with production of conventional antibodies and yields anddegree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid certain of the problems associated withantibodies that possess murine or rat variable and/or constant regions.The presence of such murine or rat derived proteins can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of human antibody functioninto a rodent so that the rodent produces fully human antibodies.

Human Antibodies

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain humanheavy chain and light chain genes within their genome. For example, aXenoMouse® mouse containing 245 kb and 190 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus is described in Green et al., Nature Genetics 7:13-21(1994). The work of Green et al. was extended to the introduction ofgreater than approximately 80% of the human antibody repertoire throughutilization of megabase-sized, germline configuration YAC fragments ofthe human heavy chain loci and kappa light chain loci, respectively. SeeMendez et al., Nature Genetics 15:146-56 (1997) and U.S. patentapplication Ser. No. 08/759,620, filed Dec. 3, 1996, the disclosures ofwhich are hereby incorporated by reference. Further, XenoMouse® micehave been generated that contain the entire lambda light chain locus(U.S. Patent Application Ser. No. 60/334,508, filed Nov. 30, 2001). And,XenoMouse® mice have been generated that produce multiple isotypes (see,e.g., WO 00/76310). XenoMouse® strains are available from Abgenix, Inc.(Fremont, Calif.).

The production of XenoMouse® mice is further discussed and delineated inU.S. patent application Ser. Nos. 07/466,008, filed Jan. 12, 1990, Ser.No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24,1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No.08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995, Ser. No.08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995,Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun.5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,and Ser. No. 08/759,620, filed Dec. 3, 1996 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoMendez et al. Nature Genetics 15:146-156 (1997) and Green and JakobovitsJ. Exp. Med., 188:483-495 (1998). See also European Patent No., EP463,151 B1, grant published Jun. 12, 1996, International PatentApplication No., WO 94/02602, published Feb. 3, 1994, InternationalPatent Application No., WO 96/34096, published Oct. 31, 1996, WO98/24893, published Jun. 11, 1998, WO 00/76310, published Dec. 21, 2000.The disclosures of each of the above-cited patents, applications, andreferences are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. Nos. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No.546,073 B1, International Patent Application Nos. WO 92/03918, WO92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No.5,981,175, the disclosures of which are hereby incorporated by referencein their entirety. See further Taylor et al., (1992), Chen et al.,(1993), Tuaillon et al., (1993), Choi et al., (1993), Lonberg et al.,(1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild etal., (1996), the disclosures of which are hereby incorporated byreference in their entirety.

Kirin has demonstrated the generation of human antibodies from mice inwhich, through microcell fusion, large pieces of chromosomes, or entirechromosomes, have been introduced. See European Patent Application Nos.773,288 and 843,961, the disclosures of which are hereby incorporated byreference.

Lidak Pharmaceuticals (now Xenorex) has also demonstrated the generationof human antibodies in SCID mice modified by injection of non-malignantmature peripheral leukocytes from a human donor. The modified miceexhibit an immune response characteristic of the human donor uponstimulation with an immunogen, which consists of the production of humanantibodies. See U.S. Pat. Nos. 5,476,996 and 5,698,767, the disclosuresof which are herein incorporated by reference.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against PTH in order to vitiate concerns and/or effects ofHAMA or HACA response.

Humanization and Display Technologies

As discussed above in connection with human antibody generation, thereare advantages to producing antibodies with reduced immunogenicity. To adegree, this can be accomplished in connection with techniques ofhumanization and display techniques using appropriate libraries. It willbe appreciated that murine antibodies or antibodies from other speciescan be humanized or primatized using techniques well known in the art.See e.g., Winter and Harris, Immunol Today 14:43-46 (1993) and Wright etal., Crit, Reviews in Immunol. 12:125-168 (1992). The antibody ofinterest may be engineered by recombinant DNA techniques to substitutethe CH1, CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190 and U.S. Pat. Nos.5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085).Also, the use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al., P.N.A.S. 84:3439 (1987) and J.Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or othercell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al., “Sequences ofProteins of Immunological Interest,” N.I.H. publication no. 91-3242(1991). Human C region genes are readily available from known clones.The choice of isotype will be guided by the desired effector functions,such as complement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′).sub.2 and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al., Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al., P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al., Cell 41:885 (1985)). Also, aswill be appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display-type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau, PNAS USA 94:4937-4942(1997) (ribosomal display), Parmley and Smith, Gene 73:305-318 (1988)(phage display), Scott, TIBS 17:241-245 (1992), Cwirla et al., PNAS USA87:6378-6382 (1990), Russel et al., Nucl. Acids Res. 21:1081-1085(1993), Hoganboom et al., Immunol. Reviews 130:43-68 (1992), Chiswelland McCafferty, TIBTECH 10:80-84 (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated against PTHexpressing cells, PTH itself, forms of PTH, epitopes or peptidesthereof, and expression libraries thereto (see e.g. U.S. Pat. No.5,703,057) which can thereafter be screened as described above for theactivities described above.

Preparation of Antibodies

Antibodies in accordance with the invention were prepared through theutilization of the XenoMouse® technology, as described below. Such mice,then, are capable of producing human immunoglobulin molecules andantibodies and are deficient in the production of murine immunoglobulinmolecules and antibodies. Technologies utilized for achieving the sameare disclosed in the patents, applications, and references disclosed inthe previous section, herein. In particular, however, a preferredembodiment of transgenic production of mice and antibodies therefrom isdisclosed in U.S. patent application Ser. No. 08/759,620, filed Dec. 3,1996 and International Patent Application Nos. WO 98/24893, publishedJun. 11, 1998 and WO 00/76310, published Dec. 21, 2000, the disclosuresof which are hereby incorporated by reference. See also Mendez et al.,Nature Genetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Antibodies, as described herein, are neutralizing high affinityantibodies to human PTH. Further, in some embodiments, the antibodiescross react with rat PTH. Several different methods have been usedhistorically to generate monoclonal antibodies or polyclonal antibodiesagainst the N-terminus of human PTH. These approaches have includedimmunizing with full length human PTH (hPTH) or bovine PTH (bPTH)(Vieira et al., Braz. J. Med. Biol. Res. 21:1005-1011 (1988)), syntheticpeptides of human PTH (1-34 or 1-37) (Visser et al., Acta Endocrinol.90:90-102 (1979)); Logue et al., J. Immunol. Methods 137:159-66 (1991)),and multiple antigenic peptides (MAP) of hPTH (1-10), hPTH (9-18) andhPTH (24-37) (Magerlein et al., Drug Res. 48:783-87 (1998)). Theseapproaches did not produce antibodies suitable for human therapeutics.(See section entitled “Therapeutic Administration and Formulation”herein for therapeutic criteria.) High affinity antibodies to HPTH aredifficult to make because of B cell tolerance to the peptide. However,Bradwell et al., (1999) have demonstrated that immunization with amixture of human PTH (1-34) and bovine PTH (1-34) MAPs followed by amixture of human and bovine MAPs targeting the hPTH (51-84) and bPTH(51-86) was effective in breaking B-cell tolerance to PTH in a humanpatient with an inoperable parathyroid tumor.

The approach described herein was designed to overcome B-cell toleranceto hPTH as well as to produce a fully human monoclonal antibody suitablefor therapeutic and diagnostic use. XenoMouse® animals were immunizedwith synthetic peptides of PTH (hPTH (1-34) and rPTH (1-34)), becausesynthetic peptides have been successfully used to generate antibodiesspecific to endogenous human PTH (Visser et al., (1979)). Furthermore,because the N-terminus of murine PTH is highly conserved with human PTH(85% identity) and rat PTH (91%), the combination of peptides was usedas an immunogen to break B-cell tolerance to murine PTH throughmolecular mimicry, thereby allowing the generation of high affinityhuman anti-human PTH antibodies. These peptides were both coupled tokeyhole limpet hemocyanin and emulsified in complete Freund's adjuvantor incomplete Freund's adjuvant to enhance the immunogenicity of theseproteins.

After immunization, lymphatic cells (such as B cells) were recoveredfrom the mice that expressed antibodies, and such recovered cell linesfused with a myeloid-type cell line to prepare immortal hybridoma celllines. Such hybridoma cell lines were screened and selected to identifyhybridoma cell lines that produced antibodies specific to the antigen ofinterest. Herein, the production of multiple hybridoma cell lines thatproduce antibodies specific to PTH is described. Further, acharacterization of the antibodies produced by such cell lines isprovided, including nucleotide and amino acid sequence analyses of theheavy and light chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells may be directly assayed. For example, the CD19+ Bcells may be isolated from hyperimmune XenoMouse® mice and allowed toproliferate and differentiate into antibody-secreting plasma cells.Antibodies from the cell supernatants are then screened by ELISA forreactivity against the PTH immunogen. The supernatants are also screenedfor immunoreactivity against fragments of PTH to further epitope map thedifferent antibodies and with rat PTH to determine speciescross-reactivity. Single plasma cells secreting antibodies with thedesired specificities are then isolated using a PTH-specific hemolyticplaque assay (Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-48(1996)). Cells targeted for lysis are preferably sheep red blood cells(SRBCs) coated with the PTH antigen. In the presence of a B cell culturecontaining plasma cells secreting the immunoglobulin of interest andcomplement, the formation of a plaque indicates specific PTH-mediatedlysis of the sheep red blood cells surrounding the plasma cell ofinterest. The single antigen-specific plasma cell in the center of theplaque can be isolated and the genetic information that encodes thespecificity of the antibody can be isolated from the single plasma cell.As an alternative to B-cell culture, antigen-specific plasma cells canbe isolated directly from CD138+ splenocytes or lymphocytes ofhyperimmune animals. Independent of the method of isolation, the DNAencoding the heavy and light chain variable regions of the antibody canbe cloned after performing reverse-transcriptase PCR. Such cloned DNAcan then be further inserted into a suitable expression vector,preferably a vector cassette such as a pcDNA, more preferably such apcDNA vector containing the constant domains of immunglobulin heavy andlight chain. The generated vector can then be transfected into hostcells, preferably CHO cells, and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences. The isolation ofmultiple single plasma cells that produce antibodies specific to PTH isdescribed below. Further, the genetic material that encodes thespecificity of the anti-PTH antibody can be isolated, introduced into asuitable expression vector that can then be transfected into host cells.

In general, antibodies produced by the above-mentioned cell linespossessed fully human IgG2 heavy chains with human kappa light chains.The antibodies possessed high affinities, typically possessing adissociation constant (K_(D)) of from about 10⁻⁶ through about 10⁻¹² M,when measured by either solid phase and solution phase. Antibodiespossessing a K_(D) from at least 10⁻⁹ M are preferred, with antibodiespossessing K_(D)'s of 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M being highly preferred.Due to the kinetics of monoclonal antibody interactions with secretedantigens, the efficiency of binding is greatly reduced when the antibodyK_(D) exceeds the concentration of the antigen. To efficiently suppresslevels of the antigen, the antibody K_(D) should be less than theantigen concentration.

Regarding the importance of affinity to therapeutic utility of anti-PTHantibodies, it will be understood that one can generate anti-PTHantibodies, for example, combinatorially, and assess such antibodies forbinding affinity. One approach that can be utilized is to take the heavychain cDNA from an antibody, prepared as described above and found tohave good affinity to PTH, and combine it with the light chain cDNA froma second antibody, prepared as described above and also found to havegood affinity to PTH, to produce a third antibody. The affinities of theresulting third antibodies can be measured as described herein and thosewith desirable dissociation constants isolated and characterized. Forexample, based on the high binding affinity of anti-PTH mAb 183 andanti-PTH mAb 168 antibodies, heavy chain cDNA from anti-PTH mAb 168 canbe linked to the light chain cDNA from anti-PTH mAb 183 and theresulting antibody can be assayed for binding. Alternatively, the lightchain of any of the antibodies described above can be used as a tool toaid in the generation of a heavy chain that when paired with the lightchain will exhibit a high affinity for PTH, or vice versa. For example,the light chain or the light chain variable region of anti-PTH mAb 183can be expressed with a library of heavy chains or heavy chain variableregions. These heavy chain variable regions in this library could beisolated from naïve animals, isolated from hyperimmune animals,generated artificially from libraries containing variable heavy chainsequences that differ in the CDR regions, or generated by any othermethods that produce diversity within the CDR regions of any heavy chainvariable region gene (such as random or directed mutagenesis). These CDRregions, and in particular CDR3, may be a significantly different lengthor sequence identity from the heavy chain initially paired with anti-PTHmAb 183. The resulting library could then be screened for high affinitybinding to PTH to generate a therapeutically relevant antibody moleculewith similar properties as anti-PTH mAb 183 (high affinity andneutralization). A similar process using the heavy chain or the heavychain variable region can be used to generate a therapeutically relevantantibody molecule with a unique light chain variable region.Furthermore, the novel heavy chain variable region, or light chainvariable region, can then be used in a similar fashion as describedabove to identify a novel light chain variable region, or heavy chainvariable region, that allows the generation of a novel antibodymolecule.

Another combinatorial approach that can be utilized is to performmutagenesis on germ line heavy and/or light chains that are demonstratedto be utilized in the antibodies in accordance with the inventiondescribed herein, particularly in the complementarity determiningregions (CDRs). The affinities of the resulting antibodies can bemeasured as described herein and those with desirable dissociationconstants isolated and characterized. Upon selection of a preferredbinder, the sequence or sequences encoding the same may be used togenerate recombinant antibodies as described above. Appropriate methodsof performing mutagenesis on an oligonucleotide are known to thoseskilled in the art and include chemical mutagenesis, for example, withsodium bisulfite, enzymatic misincorporation, and exposure to radiation.It is understood that the invention described herein encompassesantibodies with substantial identity, as defined herein, to theantibodies explicitly set forth herein, whether produced by mutagenesisor by any other means. Further, antibodies with conservative ornon-conservative amino acid substitutions, as defined herein, made inthe antibodies explicitly set forth herein, are included in embodimentsof the invention described herein.

Another combinatorial approach that can be used is to express the CDRregions, and in particular CDR3, of the antibodies described above inthe context of framework regions derived from other variable regiongenes. For example, CDR1 (GYSFTSYWIG (SEQ ID NO: 89)), CDR2(IISPGDSDTRYSPSFQG (SEQ ID NO: 90)) and CDR3 (QGDYVWGSYDS (SEQ ID NO:91)) of the heavy chain of anti-PTH mAb 183 could be expressed in thecontext of the framework regions of other heavy chain variable genes.Similarly, CDR1 (KSSQSLLDSDGKTYLY (SEQ ID NO: 92)), CDR2 (EVSNRFS (SEQID NO: 93)) and CDR3 (MPSIHLWT (SEQ ID NO: 94)) of the light chain ofanti-PTH mAb 183 could be expressed in the context of the frameworkregions of other light chain variable genes. In addition, the germlinesequences of these CDR regions could be expressed in the context ofother heavy or light chain variable region genes (for example, heavychain CDR2: IIYPGDSDTRYSPSFQG (SEQ ID NO: 95)). The resulting antibodiescan be assayed for specificity and affinity and may allow the generationof a novel antibody molecule.

Furthermore, a heavy chain CDR3 with the ability to interact with PTHfrom the germline D-region 21-10 can be created. One reading frame ofthe germline form of this D-region encodes the core binding region ofCDR3 (YYDYVWGSYAYT (SEQ ID NO: 96)) as underlined. The underlined coresequence, or fragments thereof, with random flanking amino acids couldbe expressed to generate a novel CDR3 with similar specificity to theCDR3 identified in anti-PTH mAb 183. The new heavy chain could generatean antibody with similar or improved binding properties compared to theoriginal anti-PTH mAb 183. One may also mutate this D-region encodingCDR3 to generate a functionally equivalent antibody to anti-PTH mAb 183.For example, the aspartic acid (“D”) of the sequence (DYVWGSY (SEQ IDNO: 97)) can be mutated to any other amino acid and when paired with arelevant light chain the new antibody can be assayed for specificity andhigh affinity. Similarly, amino acids encoded in the CDR3 of the lightchain can be added and/or removed by slightly altering the junction ofthe V region and the J region as well as altering the number andidentity of the nucleotides used to join these two light chain segments.

In the preferred embodiment, the properties of anti-PTH mAb 183 include,for example, high affinity of the antibody for PTH (K_(D) of 10⁻¹⁰ orbetter), specificity for a neutralizing epitope on the N-terminus ofhuman PTH or orthologous proteins, the ability to neutralize calciumflux in PTH-responsive cells, and the ability to inhibit hypercalcemiain rats infused with human PTH. The examples are illustrative of themany possible means under the current art to use the sequences of theinvention to aid in the generation of an antibody with similarproperties to anti-PTH mAb 183. Any improvements to the current art orany generation of unique antibodies through future or conventionaltechnology with the aforementioned properties ascribed to anti-PTH mAb183 are deemed to be “functionally equivalent” to anti-PTH mAb 183 andthereby are included in embodiments of the invention described herein.

As will be appreciated, antibodies in accordance with the inventiondescribed herein can be expressed in various cell lines. Sequencesencoding particular antibodies can be used for transformation of asuitable mammalian host cell. Transformation can be accomplished by anyknown method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), and a number of other cell lines. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels and produce antibodies with constitutive PTHbinding properties.

Epitope Mapping

Immunoblot Analysis

The binding of the antibodies described herein to PTH can be examined bya number of methods. For example, PTH may be subjected to SDS-PAGE andanalyzed by immunoblotting. The SDS-PAGE may be performed either in theabsence or presence of a reduction agent. Such chemical modificationsmay result in the methylation of cysteine residues. Accordingly, it ispossible to determine whether the anti-PTH antibodies described hereinbind to a linear epitope on PTH.

Antibody binding can also be analyzed by the creation of an overlappinglibrary of peptides corresponding to particular fragments of PTH. Forexample, a series of 8, 10, or 12-mer peptides can be synthesized withpeptides spanning predetermined residues (eg: residues 1-34) of the PTHamino acid sequence. Each consecutive peptide can be offset by one aminoacid from the previous one yielding a nested, overlapping library. Thena membrane carrying the 23 peptides can be probed with anti-PTH antibody183 (1 mg/ml) and detected with HRP-conjugated secondary anti-human IgGantibody using enhanced chemiluminescence (ECL).

To further refine the contact residues involved in the binding ofanti-PTH mAb 183 to PTH, an alanine-scanning mutation approach can beemployed using a custom-peptide array. In this type of study, each aminoacid in the epitope is replaced by the amino acid alanine, in order todetermine the amino acids that are necessary for antibody binding.

Diagnostic Use

Antibodies in accordance with the invention described herein are usefulfor diagnostic assays, and, particularly, in vitro assays, for example,for use in determining the level of circulating PTH in the bloodstream.It is possible to determine the presence and/or severity ofhyperparathyroidism in a subject based on expression levels of the PTHantigen. Patient samples, preferably blood, and more preferably bloodserum, are taken from subjects diagnosed as being at various stages inthe progression of hyperparathyroidism, and/or at various points in thetherapeutic treatment of the disease. The concentration of the PTHantigen present in the blood samples is determined using a method thatspecifically determines the amount of the antigen that is present. Sucha method includes an ELISA method in which, for example, antibodies ofthe invention may be conveniently immobilized on an insoluble matrix,such as a polymer matrix. Using a population of samples that providesstatistically significant results for known levels of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each level is designated.

In order to determine the degree of hyperparathyroidism in a subjectunder study, or to characterize the response of the subject to a courseof therapy, a sample of blood is taken from the subject and theconcentration of the PTH antigen present in the sample is determined.The concentration so obtained is used to identify in which range ofconcentrations the value falls. The range so identified correlates witha level of disease progression or a level of therapy identified in thevarious populations of diagnosed subjects, thereby providing a level inthe subject under study.

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay can be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

For example, antibodies, including antibody fragments, can be used toqualitatively or quantitatively detect the expression of PTH proteins.As noted above, the antibody preferably is equipped with a detectable,e.g., fluorescent label, and binding can be monitored by lightmicroscopy, flow cytometry, fluorimetry, or other techniques known inthe art. These techniques are particularly suitable if the amplifiedgene encodes a cell surface protein, e.g., a growth factor. Such bindingassays are performed as known in the art.

In situ detection of antibody binding to the PTH protein can beperformed, for example, by immunofluorescence or immunoelectronmicroscopy. For this purpose, a tissue specimen is removed from thepatient, and a labeled antibody is applied to it, preferably byoverlaying the antibody on a biological sample. This procedure alsoallows for determining the distribution of the marker gene product inthe tissue examined. It will be apparent for those skilled in the artthat a wide variety of histological methods are readily available for insitu detection.

One of the most sensitive and most flexible quantitative methods forquantitating differential gene expression is RT-PCR, which can be usedto compare mRNA levels in different sample populations, in normal andtumor tissues, with or without drug treatment, to characterize patternsof gene expression, to discriminate between closely related mRNAs, andto analyze RNA structure.

The first step is the isolation of mRNA from a target sample. Thestarting material is typically total RNA isolated from a disease tissueand corresponding normal tissues, respectively. Thus, mRNA can beextracted, for example, from frozen or archived paraffin-embedded andfixed (e.g. formalin-fixed) samples of diseased tissue for comparisonwith normal tissue of the same type. Methods for mRNA extraction arewell known in the art and are disclosed in standard textbooks ofmolecular biology, including Ausubel et al., Current Protocols ofMolecular Biology, John Wiley and Sons (1997). Methods for RNAextraction from paraffin embedded tissues are disclosed, for example, inRupp and Locker, Lab Invest., 56:A67 (1987), and De Andrés et al.,BioTechniques, 18:42044 (1995). In particular, RNA isolation can beperformed using purification kit, buffer set and protease fromcommercial manufacturers, such as Qiagen, according to themanufacturer's instructions. For example, total RNA from cells inculture can be isolated using Qiagen RNeasy mini-columns. Total RNA fromtissue samples can be isolated using RNA Stat-60 (Tel-Test).

As RNA cannot serve as a template for PCR, the first step indifferential gene expression analysis by RT-PCR is the reversetranscription of the RNA template into cDNA, followed by its exponentialamplification in a PCR reaction. The two most commonly used reversetranscriptases are avilo myeloblastosis virus reverse transcriptase(AMV-RT) and Moloney murine leukemia virus reverse transcriptase(MMLV-RT). The reverse transcription step is typically primed usingspecific primers, random hexamers, or oligo-dT primers, depending on thecircumstances and the goal of expression profiling. For example,extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit(Perkin Elmer, Calif., USA), following the manufacturer's instructions.The derived cDNA can then be used as a template in the subsequent PCRreaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ endonuclease activity. Thus,TaqMan PCR typically utilizes the 5′-nuclease activity of Taq or Tthpolymerase to hydrolyze a hybridization probe bound to its targetamplicon, but any enzyme with equivalent 5′ nuclease activity can beused. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TaqMan RT-PCR can be performed using commercially available equipments,such as, for example, ABI PRIZM 7700™ Sequence Detection System™(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In apreferred embodiment, the 5′ nuclease procedure is run on a real-timequantitative PCR device such as the ABI PRIZM 7700™ Sequence DetectionSystem™. The system consists of a thermocycler, laser, charge-coupleddevice (CCD), camera and computer. The system amplifies samples in a96-well format on a thermocycler. During amplification, laser-inducedfluorescent signal is collected in real-time through fiber optics cablesfor all 96 wells, and detected at the CCD. The system includes softwarefor running the instrument and for analyzing the data.

5′-Nuclease assay data are initially expressed as Ct, or the thresholdcycle. As discussed above, fluorescence values are recorded during everycycle and represent the amount of product amplified to that point in theamplification reaction. The point when the fluorescent signal is firstrecorded as statistically significant is the threshold cycle (Ct). TheΔCt values are used as quantitative measurement of the relative numberof starting copies of a particular target sequence in a nucleic acidsample when comparing the expression of RNA in a cell from a diseasedtissue with that from a normal cell.

To minimize errors and the effect of sample-to-sample variation, RT-PCRis usually performed using an internal standard. The ideal internalstandard is expressed at a constant level among different tissues, andis unaffected by the experimental treatment. RNAs most frequently usedto normalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin.

Differential gene expression can also be identified, or confirmed usingthe microarray technique. In this method, nucleotide sequences ofinterest are plated, or arrayed, on a microchip substrate. The arrayedsequences are then hybridized with specific DNA probes from cells ortissues of interest.

In a specific embodiment of the microarray technique, PCR amplifiedinserts of cDNA clones are applied to a substrate in a dense array.Preferably at least 10,000 nucleotide sequences are applied to thesubstrate. The microarrayed genes, immobilized on the microchip at10,000 elements each, are suitable for hybridization under stringentconditions. Fluorescently labeled cDNA probes may be generated throughincorporation of fluorescent nucleotides by reverse transcription of RNAextracted from tissues of interest. Labeled cDNA probes applied to thechip selectively hybridize to each spot of DNA on the array. Afterstringent washing to remove non-specifically bound probes, the chip isscanned by confocal laser microscopy. Quantitation of hybridization ofeach arrayed element allows for assessment of corresponding mRNAabundance. With dual color fluorescence, separately labeled cDNA probesgenerated from two sources of RNA are hybridized pairwise to the array.The relative abundance of the transcripts from the two sourcescorresponding to each specified gene is thus determined simultaneously.The miniaturized scale of the hybridization affords a convenient andrapid evaluation of the expression pattern for large numbers of genes.Such methods have been shown to have the sensitivity required to detectrare transcripts, which are expressed at a few copies per cell, and toreproducibly detect at least approximately two-fold differences in theexpression levels (Schena et al., Proc. Natl. Acad. Sci. USA,93(20)L106-49). The methodology of hybridization of nucleic acids andmicroarray technology is well known in the art.

Additional Criteria for Antibody Therapeutics

As discussed herein, the function of the PTH antibody was found to beimportant to at least a portion of its mode of operation. By function,is meant, by way of example, the activity of the anti-PTH antibody inbinding to PTH. Accordingly, in certain respects, it may be desirable inconnection with the generation of antibodies as therapeutic candidatesagainst PTH that the antibodies be capable of fixing complement andparticipating in complement-dependent cytotoxicity (CDC). For example,the anti-PTH antibodies according to embodiments of the inventiondescribed herein may be made capable of effector function, including CDCand antibody-dependent cellular cytotoxicity (ADCC). There are a numberof isotypes of antibodies that are capable of the same, including,without limitation, the following: murine IgM, murine IgG2a, murineIgG2b, murine IgG3, human IgM, human IgG1, and human IgG3. It will beappreciated that antibodies that are generated need not initiallypossess such an isotype but, rather, the antibody as generated canpossess any isotype and the antibody can be isotype switched thereafterusing conventional techniques that are well known in the art. Suchtechniques include the use of direct recombinant techniques (see, e.g.,U.S. Pat. Nos. 4,816,397 and 6,331,415), cell-cell fusion techniques(see, e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418), among others.

In the cell-cell fusion technique, a myeloma or other cell line isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

By way of example, certain anti-PTH antibodies of the inventiondescribed herein are human anti-PTH IgG2 antibodies. If such an antibodypossessed desired binding to the PTH molecule, it could be readilyisotype switched to generate a human IgM, human IgG1, or human IgG3isotype, while still possessing the same variable region (which definesthe antibody's specificity and some of its affinity).

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with certain alternate “functional” attributes through isotypeswitching.

Design and Generation of Other Therapeutics

In accordance with embodiments of the invention described herein andbased on the activity of the antibodies that are produced andcharacterized herein with respect to PTH, the design of othertherapeutic modalities beyond antibody moieties is facilitated. Suchmodalities include, without limitation, advanced antibody therapeutics,such as bispecific antibodies, immunotoxins, and radiolabeledtherapeutics, generation of peptide therapeutics, gene therapies,particularly intrabodies, antisense therapeutics, and small molecules.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecifics, immunotoxins, or radiolabels, for example.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to PTH and another to a second molecule that are conjugatedtogether, (ii) a single antibody that has one chain specific to PTH anda second chain specific to a second molecule, or (iii) a single chainantibody that has specificity to PTH and the other molecule. Suchbispecific antibodies can be generated using techniques that are wellknown for example, in connection with (i) and (ii) see e.g., Fanger etal. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra. and inconnection with (iii) see e.g., Traunecker et al. Int. J. Cancer (Suppl)7:51-52 (1992). In each case, the second specificity can be made to theheavy chain activation receptors, including, without limitation, CD16 orCD64 (see e.g., Deo et al. 18:127 (1997)) or CD89 (see e.g., Valerius etal. Blood 90:4485-4492 (1997)).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902.

Therapeutic Administration and Formulations

Biologically active anti-PTH antibodies in accordance with the inventiondescribed herein may be used in a sterile pharmaceutical preparation orformulation to reduce the level of serum PTH thereby effectivelytreating pathological conditions where, for example, serum PTH isabnormally elevated. Such conditions include, for instance,hyperparathyroidism, such as primary, secondary, and tertiaryhyperparathyroidism, hypercalcemia, and hypophosphatemia (if normalrenal function) or hyperphosphatemia (if impaired renal function). Theanti-PTH antibody preferably possesses adequate affinity to potentlysuppress PTH to within the target therapeutic range, and preferably hasan adequate duration of action to allow for infrequent dosing. In thetreatment of secondary hyperparathyroidism in hemodialysis patients, PTHshould preferably be suppressed by the administration of the antibodywith a minimum duration of at least two days to provide continuousefficacy between dialysis sessions, which occur three times weekly. Suchduration of action allows for intravenous dosing at the time ofhemodialysis, which will result in increased compliance and convenienceto patients and health care providers. In other hyperparathyroid patientpopulations, a prolonged duration of action of greater than two days,preferably three to five days, more preferably seven to ten days, willallow for less frequent and more convenient dosing schedules byalternate parenteral routes such as subcutaneous or intramuscularinjection.

The preference for high affinity and prolonged duration of actionextends to all conditions of hyperparathyroidism, such as primaryhyperparathyroidism, secondary hyperparathyroidism and tertiaryhyperparathyroidism. A convenient prolonged duration of action is onethat preferably exceeds two days, preferably one that exceeds five toseven days, more preferably one that exceeds ten days, following asingle dose of the antibody. A preferred antibody of the invention has aK_(D) of 10⁻¹⁰ M, a half-life exceeding 1.5 days, preferably 2 to 4days, more preferably 6 to 10 days and suppresses the levels ofcirculating PTH by greater than 50%, preferably by greater than 60%,65%, or 70%, more preferably by greater than 75%, 80% or 85%. In apreferred embodiment, an antibody demonstrating a suppression of greaterthan 75% for greater than 1.5 days is provided.

Biologically active anti-PTH antibodies of the instant invention may beemployed alone or in combination with other therapeutic agents. Forexample, current approved therapy for secondary hyperparathyroidism dueto chronic renal failure (CRF) consists of (1) calcitriol (active formof vitamin D) and analogs, which act to promote absorption of calcium inthe gastrointestinal tract, (2) calcium carbonate with meals to bind tophosphate and prevent its absorption in the gut, (3) other phosphatebinders, and (4) calcimimetics. One of the main causes of secondaryhyperparathyroidism in CRF is high levels of serum phosphate actingdirectly on the parathyroid glands causing them to produce PTH.Phosphate levels increase due to inadequate phosphate excretion due tothe kidney failure. The above-mentioned current treatments act toprevent bone resorption and could be used in combination with antibodiesof the invention described herein in a therapeutic regime.

When used for in vivo administration, the antibody formulation should besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes, prior to or followinglyophilization and reconstitution. The antibody ordinarily will bestored in lyophilized form or in solution. Therapeutic antibodycompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having anadapter that allows retrieval of the formulation, such as a stopperpierceable by a hypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, subcutaneous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, intrathecal, inhalation or intralesional routes, or bysustained release systems as noted below. The antibody is preferablyadministered continuously by infusion, by bolus injection, or bysubcutaneous injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

Antibodies in accordance with the invention may be prepared in a mixturewith a pharmaceutically acceptable carrier. This therapeutic compositioncan be administered intravenously or through the nose or lung,preferably as a liquid or powder aerosol (lyophilized). The compositionmay also be administered parenterally or subcutaneously as desired. Whenadministered systemically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art.

Briefly, dosage formulations of the compounds of the invention describedherein are prepared for storage or administration by mixing the compoundhaving the desired degree of purity with physiologically acceptablecarriers, excipients, or stabilizers. Such materials are non-toxic tothe recipients at the dosages and concentrations employed, and includebuffers such as TRIS HCl, phosphate, citrate, acetate and other organicacid salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) peptides such as polyarginine, proteins,such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidinone; amino acids such as glycine, glutamicacid, aspartic acid, or arginine; monosaccharides, disaccharides, andother carbohydrates including cellulose or its derivatives, glucose,mannose, or dextrins; chelating agents such as EDTA; sugar alcohols suchas mannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington'sPharmaceutical Sciences (18th ed., Mack Publishing Company, Easton, Pa.1990). For example, dissolution or suspension of the active compound ina vehicle such as water or naturally occurring vegetable oil likesesame, peanut, or cottonseed oil or a synthetic fatty vehicle likeethyl oleate or the like may be desired. Buffers, preservatives,antioxidants and the like can be incorporated according to acceptedpharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., 15:167-277 (1981) andLanger, Chem. Tech., 12:98-105 (1982), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-56 (1983)), non-degradable ethylene-vinyl acetate (Langer et al.,supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-release compositions also include liposomally entrappedantibodies of the invention. Liposomes containing such antibodies areprepared by methods known per se: U.S. Pat. No. DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. USA, 82:3688-92 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA, 77:4030-34 (1980); EP 52,322; EP 36,676; EP88,046; EP 143,949; 142,641; Japanese patent application 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages may be determined by either in vitro or in vivomethods.

An effective amount of the antibody of the invention to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 0.001 mg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Desirable dosage concentrations include 0.001 mg/kg,0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, and 100 mg/kg or more.Typically, the clinician will administer the therapeutic antibody untila dosage is reached that achieves the desired effect. The progress ofthis therapy is easily monitored by conventional assays or as describedherein.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the invention described herein.

Example 1 PTH Antigen Preparation

The following PTH peptides were used in the experiments describedherein.

Human PTH (1-84): (SEQ ID NO: 81)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ Human PTH (1-34): (SEQ ID NO: 82)SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF Human PTH (7-84): (SEQ ID NO: 83)LMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ Human PTH (18-48): (SEQ ID NO: 84)MERVEWLRKKLQDVHNFVALGAPLAPRDAGS Human PTH-related peptide (1-34): (SEQID NO: 85) AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA Rat PTH (1-84): (SEQ IDNO: 86) AVSEIQLMHNLGKHLASVERMQWLRKKLQDVHNFVSLGVQMAAREGSYQRPTKKEENVLVDGNSKSLGEGDKADVDVLVKAKSQ Rat PTH (1-34): (SEQ ID NO: 87)AVSEIQLMHNLGKHLASVERMQWLRKKLQDVHNF Cynomolgus PTH (1-84): (SEQ ID NO:88) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFIALGAPLAPRDAGSQRPRKKEDNILVESHEKSLGEADKADVDVLTKAKSQ.

These peptides were purchased from Bachem California Inc., Torrance,Calif. Cynomolgus PTH (1-84) was also purchased from CS Bio Company,Inc., San Carlos, Calif.

Antigen Preparation. The antigen used for the immunization of XenoMouse®animals was prepared as follows. Human PTH1-34 (500 mcg) was mixed withrat PTH1-34 (500 mcg) and dissolved in 500 mcL of conjugation buffer(0.1M MES, 0.9M NaCl, pH 4.7). Two milligrams of keyhole limpethemocyanin (KLH; Pierce, Rockford, Ill.) were dissolved in 200 mcL ofdistilled water and then added to the PTH mixture. PTH and KLH werecross-linked by the addition of 50 mcL of a 10 mg/mL stock solution of1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC,Pierce, Rockford, Ill.) and incubation of the mixture for 2 hours atroom temperature. The unreacted EDC was removed by overnight dialysisthrough a 1 kDa cutoff membrane against PBS pH 7.4.

Example 2 Anti-PTH Antibodies

Antibody Generation

Immunization of animals. Monoclonal antibody against PTH was developedby sequentially immunizing XenoMouse® mice (XenoMouse® XG2, Abgenix,Inc. Fremont, Calif.). Synthetic 34mer human and rat PTH (50/50) coupledto KLH as described above was used as the antigen. The initialimmunization was with 10 μg of antigen mixed 1:1 v/v with CompleteFreund's Adjuvant (CFA, Sigma, Oakville, ON) per mouse. Subsequentboosts were made first with 10 μg of antigen admixed 1:1 v/v withIncomplete Freund's Adjuvant (IFA, Sigma, Oakville, ON) per mouse,followed by three injections with 10 μg of antigen mixed 1:1 v/v withIFA, and then a final boost of 10 μg of antigen admixed 1:1 v/v with IFAper mouse. In particular, each mouse was immunized at the base of thetail by subcutaneous injection. The animals were immunized on days 0,14, 28, 42 and 54. The animals were bled on day 49 to obtain sera forharvest selection as described below.

Selection of animals for harvest. Anti-PTH antibody titers weredetermined by ELISA. PTH (84mer; 1 μg/mL) was coated onto Costar LabcoatUniversal Binding Polystyrene 96-well plates (Corning, Acton, Mass.)overnight at four degrees. The solution containing unbound PTH wasremoved and the plates were treated with UV light (365 nm) for 4 minutes(4000 microjoules). The plates were washed five times with dH₂O.XenoMouse® sera from the PTH immunized animals, or naïve XenoMouse®animals, were titrated in 2% milk/PBS at 1:2 dilutions in duplicate froma 1:100 initial dilution. The last well was left blank. The plates werewashed five times with dH₂O. A goat anti-human IgG Fc-specifichorseradish peroxidase (HRP, Pierce, Rockford, Ill.) conjugated antibodywas added at a final concentration of 1 μg/mL for 1 hour at roomtemperature. The plates were washed five times with dH₂O. The plateswere developed with the addition of TMB chromogenic substrate(Gaithersburg, Md.) for 30 minutes and the ELISA was stopped by theaddition of 1 M phosphoric acid. The specific titer of individualXenoMouse® animals was determined from the optical density at 450 nm andare shown in Table 2. The titer represents the reciprocal dilution ofthe serum and therefore the higher the number the greater the humoralimmune response to PTH.

TABLE 2 Mouse I.D. Titer M469-1 16000 M469-2 4000 M469-3 16000 M469-432000 M469-5 8000 M469-6 32000 M469-7 8000 M469-8 32000 M469-9 >32000M469-10 3200 Naïve 1000

XenoMouse® animals (M469-4, M469-6, M469-8 and M469-9) were selected forharvest based on the serology data in Table 2.

Culture and selection of B cells. B cells from the harvested animalswere cultured and those secreting PTH-specific antibodies were isolatedas described in Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-48(1996). ELISA, performed as described above, was used to identifyprimary PTH-specific wells. One hundred plates cultured at 500, 250, 125or 50 cells/well were screened on PTH to identify the antigen-specificwells and 98 wells showed ODs significantly over background (0.1), arepresentative sample of which are shown in Table 3.

TABLE 3 Positives above cutoff OD of: Tissue 0.1 0.2 0.3 0.4 0.5 0.6 0.70.8 0.9 1.0 1.5 2.0 Pooled LN @ 500 cells/well 861 133 52 30 23 16 11 98 6 1 1 (plates #279-303) Pooled LN @ 250 cells/well 436 90 34 23 17 138 7 6 5 1 0 (plates #304-328) Pooled LN @ 125 cells/well 32 8 7 3 3 1 11 1 1 0 0 (plates #329-353) Pooled LN @ 50 cells/well 31 7 5 5 2 2 1 1 10 0 0 (plates #354-378) Total Number Positives: 1360 238 98 61 45 32 2118 16 12 2 1

These data indicated a very low frequency of hits and indicated that thewells were monoclonal for antigen-specificity at all cell dilutions.These 98 positive wells were rescreened on antigen (Table 4) and only 48wells were found to repeat as clearly antigen-specific wells. These 48wells were analyzed for binding to either hPTH7-84 or hPTH18-48.Antibodies specific to hPTH7-34 were desired as they were unlikely tocross-react with human PTHrp. A total of 14 wells with reactivity foreither hPTH7-84 or hPTH18-48 were identified and these wells weresubsequently analyzed for relative affinity using into limiting antigenELISA analysis as described below. The well 292A10 was the source ofanti-PTH mAb 183. This well bound nicely to human PTH 1-84 (OD 1.4),human PTH 7-84 (OD 1.95), human PTH 18-48 (OD 3.26) and rat PTH 1-84 (OD0.66). These data were qualitative and were useful to indicate that thisantibody binds PTH between amino acids 18 and 34 (as the immunogen wasonly the N-terminal fragment 1-34) and that this antibody was able tobind to rat PTH.

Limiting antigen analysis. The limiting antigen analysis is a methodthat affinity ranks the antigen-specific antibodies in the B cellculture supernatants relative to all other antigen-specific antibodies.The concept being that in the presence of a very low coating of antigenthat only the highest affinity antibodies will be able to bind to anydetectable level at equilibrium. (See, e.g., U.S. Ser. No. 60/337,250,filed Dec. 3, 2001.) Biotinylated PTH was bound to streptavidin platesat 25 ng/mL for 30 minutes at room temperature on 96-well cultureplates. Each plate was washed 5 times with dH₂O, before 40 μL of 1% milkin PBS with 0.05% sodium azide were added to the plate, followed by 10μL of B cell supernatant added to each well. After 18 hours at roomtemperature on a shaker, the plates were again washed 5 times with dH₂O.To each well was added 50 μL of goat (Gt) anti-Human (Fc)-HRP at 1μg/mL. After 1 hour at room temperature, the plates were again washed 5times with dH₂O and 50 μL of tetramethylbenzidine (TMB) substrate wereadded to each well. The reaction was stopped by the addition of 50 μL of1M phosphoric acid to each well and the plates were read at wavelength450 nm to give the results shown in Table 4.

Quantitation of antigen-specific antibody in the B-cell culturesupernatants have indicated the concentration ranges from 10 ng/mL to500 ng/mL. As it is difficult to determine the concentration ofantigen-specific antibody in every antigen-specific well, the resultsgenerated from limiting antigen analysis was compared to a titration ofrecombinant antibodies at comparable concentrations (2 ng/mL to 100ng/mL). In this assay less than half of the antibodies were able to givedetectable binding and well 292A10 (anti-PTH mAb 183) was clearlysuperior as measured by O.D. to the other culture supernatants andrecombinant antibodies at all concentrations (Table 4).

TABLE 4 Limited 1′ Hu Rat Plate ID Ag OD OD 84mer 7-84mer 18-48mer 84mer292A10 2.747 0.992 1.40 1.95 3.26 0.62 (XG2-183) 302A7 1.376 0.317 0.350.36 2.66 0.19 (XG2-168) 361B2 0.747 0.416 0.48 0.37 0.26 0.49 331H60.322 0.312 0.52 0.45 2.40 0.24 287E7 0.261 0.682 0.71 0.13 0.36 1.03315D8 0.221 0.441 0.14 0.17 0.29 0.31 279E6 0.213 0.379 0.31 0.10 0.170.19 313D5 0.170 0.664 0.12 0.29 0.43 0.30 339F5 0.120 0.319 0.40 0.210.11 0.25 279D2 0.114 0.353 0.31 0.11 0.27 0.18 307H1 0.084 0.401 0.100.14 0.30 0.42 308A1 0.079 0.312 0.19 0.22 0.30 0.45 284D9 ND 0.585 1.460.07 0.25 2.11 (XG2-262) 322F2 ND 1.87 1.01 0.15 0.34 1.41

PTH-specific Hemolytic Plaque Assay. A number of specialized reagentswere needed to conduct the assay. These reagents were prepared asfollows.

Biotinylation of Sheep red blood cells (SRBC). SRBC were stored in RPMImedia as a 25% stock. A 250 μL SRBC packed-cell pellet was obtained byaliquoting 1.0 mL of the stock into a 15-mL falcon tube, spinning downthe cells and removing the supernatant. The cell pellet was thenre-suspended in 4.75 mL PBS at pH 8.6 in a 50 mL tube. In a separate 50mL tube, 2.5 mg of Sulfo-NHS biotin was added to 45 mL of PBS at pH 8.6.Once the biotin had completely dissolved, 5 mL of SRBCs were added andthe tube rotated at RT for 1 hour. The SRBCs were centrifuged at 3000 gfor 5 min, the supernatant drawn off and 25 mL PBS at pH 7.4 as a wash.The wash cycle was repeated 3 times, then 4.75 mL immune cell media(RPMI 1640 with 10% FCS) was added to the 250 μL biotinylated-SRBC(B-SRBC) pellet to gently re-suspend the B-SRBC (5% B-SRBC stock). Stockwas stored at 4° C. until needed.

Streptavidin (SA) coating of B-SRBC. One mL of the 5% B-SRBC stock wastransferred into to a fresh eppendorf tube. The B-SRBC cells werepelleted with a pulse spin at 8000 rpm (6800 rcf) in microfuge, thesupernatant drawn off, the pellet re-suspended in 1.0 mL PBS at pH 7.4,and the centrifugation repeated. The wash cycle was repeated 2 times,then the B-SRBC pellet was resuspended in 1.0 mL of PBS at pH 7.4 togive a final concentration of 5% (v/v). 10 μL of a 10 mg/mL streptavidin(CalBiochem, San Diego, Calif.) stock solution was added and the tubemixed and rotated at RT for 20 min. The washing steps were repeated andthe SA-SRBC were re-suspended in 1 μmL PBS pH 7.4 (5% (v/v)).

Human PTH 1-34 coating of SA-SRBC. The SA-SRBC were coated withbiotinylated-Human PTH 1-34 at 10 μg/mL, then mixed and rotated at RTfor 20 min. The SRBC were washed twice with 1.0 mL of PBS at pH 7.4 asabove. The PTH-coated SRBC were re-suspended in RPMI (10% FCS) to afinal concentration of 5% (v/v).

Determination of the quality of PTH-SRBC by immunofluorescence (IF). 10μL of 5% SA-SRBC and 10 μL of 5% PTH-coated SRBC were each added toseparate fresh 1.5 mL eppendorf tube containing 40 μL of PBS. A controlhuman anti-PTH antibody was added to each sample of SRBCs at 45 μg/mL.The tubes were rotated at RT for 25 min, and the cells were then washedthree times with 100 μL of PBS. The cells were re-suspended in 50 μL ofPBS and incubated with 2 mcg/mL goat (Gt)-anti Human IgG Fc antibodyconjugated to Alexa488 (Molecular Probes, Eugene, Oreg.). The tubes wererotated at RT for 25 min, and then washed with 100 μL PBS and the cellsre-suspended in 10 μL PBS. 10 μL of the stained cells were spotted ontoa clean glass microscope slide, covered with a glass coverslip, observedunder fluorescent light, and scored on an arbitrary scale of 0-4.

Preparation of plasma cells. The contents of a single microculture wellpreviously identified by various assays as containing a B cell clonesecreting the immunoglobulin of interest were harvested. Using a100-1000 μL pipettman, the contents of the well were recovered by adding37° C. RPMI (+10% FCS). The cells were re-suspended by pipetting andthen transferred to a fresh 1.5 mL eppendorf tube (final volumeapproximately 500-700 μL). The cells were centrifuged in a microfuge at1500 rpm (240 rcf) for 2 minutes at room temperature, then the tuberotated 180 degrees and spun again for 2 minutes at 1500 rpm. The freezemedia was drawn off and the immune cells resuspended in 100 μL RPMI (10%FCS), then centrifuged. This washing with RPMI (10% FCS) was repeatedand the cells re-suspended in 60 μL RPMI (FCS) and stored on ice untilready to use.

Plaque assay. Glass slides (2×3 inch) were prepared in advance withsilicone edges and allowed to cure overnight at RT. Before use theslides were treated with approx. 5 μL of SigmaCoat (Sigma, Oakville, ON)wiped evenly over glass surface, allowed to dry and then wipedvigorously. To a 60 μL sample of cells was added 60 μL each ofPTH-coated SRBC (5% v/v stock), 4× guina pig complement (Sigma,Oakville, ON) stock prepared in RPMI (FCS), and 4× enhancing sera stock(1:900 in RPMI (FCS)). The mixture (3-5 ul) was spotted onto theprepared slides and the spots covered with undiluted paraffin oil. Theslides were incubated at 37° C. for a minimum of 45 minutes.

Plaque assay results. The coating of the sheep red blood cells withHuman PTH 1-34 worked very well. The coating was determinedqualitatively by immunofluorescent microscopy to be very high (4/4)using a control human anti-PTH antibody to detect coating compared to asecondary detection reagent alone (0/4). There was no signal detectedusing a control human anti-PTH antibody on red blood cells that wereonly coated with streptavidin (0/4). These red blood cells were thenused to identify antigen-specific plasma cells from the well 292A10 (seeTable 5). After micromanipulation to rescue the antigen-specific plasmacells, the genes encoding the variable region genes were rescued byRT-PCR on a single plasma cell.

TABLE 5 Plate ID Single Cell Numbers 292A10 PTH-SCX-179-190 302A7PTH-SCX-164-178

Expression, Purification and Characterization of Anti-PTH mAb 183

Expression. After isolation of the single plasma cells, mRNA wasextracted and reverse transcriptase PCR was conducted to generate cDNA.The cDNA encoding the variable heavy and light chains was specificallyamplified using polymerase chain reaction. The variable heavy chainregion was cloned into an IgG2 expression vector. This vector wasgenerated by cloning the constant domain of human IgG2 into the multiplecloning site of pcDNA3.1+/Hygro (Invitrogen, Burlington, ON). Thevariable light chain region was cloned into an IgK expression vector.This vector was generated by cloning the constant domain of human IgKinto the multiple cloning site of pcDNA3.1+/Neo (Invitrogen, Burlington,ON). The heavy chain and the light chain expression vectors were thenco-lipofected into a 60 mm dish of 70% confluent human embryonal kidney293 cells and the transfected cells were allowed to secrete arecombinant antibody with the identical specificity as the originalplasma cell for 24 hours. The supernatant (3 mL) was harvested from theHEK 293 cells and the secretion of an intact antibody (anti-PTH mAb 183)was demonstrated with a sandwich ELISA to specifically detect human IgG(Table 7). The specificity of anti-PTH mAb 183 was assessed throughbinding of the recombinant antibody to PTH using ELISA (Table 6). Theability of this antibody to bind to Rat PTH was also demonstrated usingan ELISA method (Table 7) as measured by O.D.

TABLE 6 Clone Cell # Secretion Binding 292A10 SC-PTH-XG2-183 >1:64 1:16

TABLE 7 Anti-PTH Conc. mAb 183 Neat 3.291 3.432 ½ 3.266 3.384 ¼ 3.3973.456 ⅛ 3.123 3.272 1/16 2.722 3.006 1/32 2.569 2.691 1/64 1.893 2.362Blank 0.215 0.182

The secretion ELISA tests were performed as follows. Control plates werecoated with 2 mg/mL Goat anti-human IgG H+ L O/N as for binding plates.Human or rat PTH (84mer; 1 μg/mL) was coated onto Costar LabcoatUniversal Binding Polystyrene 96 well plates and held overnight at fourdegrees. The plates were washed five times with dH₂O. Recombinantantibodies were titrated 1:2 for 7 wells from the undilutedminilipofection supernatant. The plates were washed five times withdH₂O. A goat anti-human IgG Fc-specific HRP-conjugated antibody wasadded at a final concentration of 1 μg/mL for 1 hour at RT for thesecretion and the two binding assays. The plates were washed five timeswith dH₂O. The plates were developed with the addition of TMB for 30minutes and the ELISA was stopped by the addition of 1 M phosphoricacid. Each ELISA plate was analyzed to determine the optical density ofeach well at 450 nm.

Purification of anti-PTH mAb 183. For larger scale production ofanti-PTH mAb 183, the heavy and light chain expression vectors (2.5 μgof each chain/dish) were lipofected into ten 100 mm dishes that were 70%confluent with HEK 293 cells. The transfected cells were incubated at37° C. for 4 days, the supernatant (6 mL) was harvested and replacedwith 6 mL of fresh media. At day 7, the supernatant was removed andpooled with the initial harvest (120 mL total from 10 plates). Theanti-PTH mAb 183 antibody was purified from the supernatant using aProtein-A Sepharose (Amersham Biosciences, Piscataway, N.J.) affinitychromatography (1 mL). The antibody was eluted from the Protein-A columnwith 500 mcL of 0.1 M Glycine pH 2.5. The eluate was dialysed in PBS pH7.4 and filter sterilized. The antibody was analyzed by non-reducingSDS-PAGE to assess purity and yield.

Deposits of plasmid DNA encoding for the heavy chain and the light chainof anti-PTH mAb 183 have been made, under Budapest Treaty conditions atthe American Type Culture Collection, 1080 University Blvd., Manassas,Va. 20110-2209, under ATCC deposit number PTA-4311 for the heavy chainand PTA-4310 for the light chain. The deposit of material does notconstitute an admission that the written description herein contained isinadequate to enable the practice of any embodiment of the invention,including the best mode thereof, nor is it to be construed as limitingthe scope of the claims to the specific illustrations that itrepresents. All restrictions on access to the deposits will be removedas required by applicable law and/or regulations.

Failure of anti-PTH mAb 183 to bind to PTH-related peptide (1-34). Theability of anti-PTH mAb 183 to bind to PTH-related protein (PTHrP) wasassessed indirectly through its ability to bind to human PTH 1-34 in thepresence of saturating levels of PTH-related peptide over a 3-logtitration. Biotinylated human PTH 1-34 was bound to streptavidin platesat 1 μg/mL for 30 minutes at RT. The plates were washed 5 times withdH2O. Anti-PTH mAb 183 was then titrated 1:2 from 1 μg/mL in thepresence or absence of 5 μg/mL PTH-related protein 1-34. The titratedantibody was then transferred to the plate coated with human PTH 1-34and allowed to bind for 1 hour at RT. After washing the plates 5 timeswith dH2O, 50 μL of Gt anti-Human (Fc)-HRP at 1 μg/mL was added to eachwell and the plates held 1 hour at RT. After washing the plates 5 timeswith dH2O, 50 μL of TMB substrate was added to each well. To stop thereaction, 50 μL of 1M phosphoric acid was added to each well. The plateswere read at wavelength 450 nm. There was no inhibition of anti-PTH mAb183 binding to human PTH 1-34 even at 1 ng/mL anti-PTH mAb 183 in thepresence of 5000 ng/mL PTHrp as shown in Table 8.

TABLE 8 Dilution of anti-PTH mAb 183 1 500 μg/ ng/ 250 125 62.5 31.315.6 mL mL ng/mL ng/mL ng/mL ng/mL ng/mL +PTHrp 2.248 2.184 2.110 2.0261.908 1.903 1.788 (5 μg/mL) 2.298 2.229 2.095 2.012 1.968 1.950 1.753−PTHrp 2.269 2.205 2.063 2.067 1.946 1.854 1.848 2.342 2.188 2.105 2.0191.975 1.880 1.715 7.8 ng/mL 3.9 ng/mL 2.0 ng/mL 1 ng/mL Blank +PTHrp1.600 1.251 0.860 0.542 0.077 (5 μg/mL) 1.598 1.316 0.900 0.583 0.087−PTHrp 1.461 1.115 0.800 0.496 0.069 1.508 1.203 0.758 0.500 0.077

Kinetic analysis. The kinetic measurements of the anti-PTH antibody(anti-PTH mAb 183) were evaluated using the KinExA technology. Thismethod involves solution-based determination of formal affinitymeasurements at equilibrium. Dual curve analysis with two known antibodyconcentrations and unknown antigen concentration was used to determinethe K_(D) measurements on human 34mer, human 84mer, cynomolgus 84mer andrat 84mer. The K_(D) was determined to be approximately 10-30 pM forsynthetic human PTH (1-84 or 1-34) and cynomolgus PTH (1-84) (CS BioCompany, Inc., San Carlos, Calif.) at room temperature. The K_(D) ofanti-PTH mAb 183 was lower (3000 pM) for synthetic rat PTH 1-84 at roomtemperature (Table 9).

The affinity of anti-PTH mAb 183 was also determined for human PTH inpooled serum from hemodialysis patients with end stage renal disease.Binding affinity was determined using an immunoassay-based Scatchardanalysis. The binding affinity for endogenous human PTH was 60 pM,consistent with the high affinity observed for synthetic human PTH usingKinExA technology.

The affinity (K_(D)) of other PTH-specific monoclonal antibodies wasalso determined. These antibodies were found to be significantly loweraffinity (350-5000 pM) than anti-PTH mAb 183 (80 pM) as determined usingBiaCore (Amersham Pharmacia) technology. The majority of theserecombinant antibodies were also found to map to a similar region orepitope on PTH contained within amino acids (18-34) as anti-PTH mAb 183(Table 10). This region or epitope has been shown to be involved inreceptor binding. (Duvos et al., Bone, (1995) 17:403-406).

TABLE 9 Antigen K_(D) K_(D) High K_(D) Low Human PTH(1-84) 22 pM 39 pM12 pM Human PTH(1-34) 33 pM 65 pM 14 pM Cynomolgus PTH (1-84) 10 pM 18pM  5 pM Rat PTH(1-84)  3 nM  5 nM  2 nM

TABLE 10 K_(D) @ K_(D) @ 25° C. 37° C. Anti-PTH (nM) - (nM) - K_(D) @37° C. Recombinant Well ID mAb ID 34mer 34mer (nM) - 84mer Bin 133A8 0111.0 3.5 ND 18-34 126B1 026 3.0 9.5 ND 18-34 123B12 045 0.5 2.0 ND 18-34119G2 057 1.0 1.0 ND 18-34 133D2 086 4.7 16.7 ND 18-34 135H11 124 0.53.3 ND 18-34 132G12 140 0.8 5.2 ND 18-34 130A1 163 3.0 7.9 41.1  18-34302A7 168 2.7 2.6 9.9 18-34 NA 168g2/ 0.28 0.64 ND ND 183k 292A10 1830.08 0.1 1.0 18-34 267D10 214 0.8 0.4 1.6 1-7 275A4 225 0.35 0.6 7.1 1-7264E5 238 0.6 0.2 3.9 1-7 284D9 262 ND ND ND 18-34 252G11 275 0.2 ND ND1-7 130C6 302 ND ND ND  1-34

The kinetics for the interactions between mAb sc275 (listed above inTable 10 as mAb 275) and PTH were studied. As indicated in Table 10, mAbsc275 bound to the first seven amino acids of human PTH. Binding of PTHto mAb sc275 was studied at 0, 2.1, 6.2, 19, 56, 167, 500, and 1500 nMPTH, at pH 7.4, and at 0, 6.2, 19, 56, 167, 500, and 1500 nM PTH, at pH6.0. Each antigen concentration was injected in duplicate. Associationswere monitored for one minute and dissociations were monitored for fiveminutes and ten minutes for the pH 7.4 and 6.0 assay, respectively. Thesurfaces were regenerated with a 12-second pulse of 50 mM H₃PO₄.

Finally, kinetic rate constants were determined by double referencingeach data set to fit globally to a 1:1 interaction model. The pH 7.4overlays of the obtained responses with the fits are shown in Table 11below.

TABLE 11 pH 7.4 37 degree C. Immobilization Density (RU) k_(a) (M⁻¹s⁻¹)k_(d) (s⁻¹) K_(D) (nM) amine 1200 3.5 × 10⁵ 8.6 × 10⁻⁴ 2.5 aldehyde 10006.1 × 10⁵ 1.2 × 10⁻³ 2.0 Fc-specific 750 4.8 × 10⁵ 9.3 × 10⁻⁴ 1.9capture

Additional kinetic constants can be calculated from BiaCore data usingthe methods described in their product literature. A binding speedconstant (k_(a)) is the value that represents strength (extent) ofbinding of an antibody with target antigen as calculated based onantigen-antibody reaction kinetics. A dissociation speed constant(k_(d)) is the value that represents the strength (extent) ofdissociation of this monoclonal antibody from target antigen ascalculated based on antigen-antibody reaction kinetics. The dissociationconstant (K_(D)) is the value obtained by dividing the dissociationspeed constant (k_(d)) value from the binding speed constant (k_(a)).These constants were used as indicators that represent affinity ofantibodies for antigen and neutralizing activity of antigen. Values fork_(a) and k_(d) for the antibodies shown in Table 10 were calculated andare given in Table 12.

TABLE 12 AB-PTH- ka @ 25° C. kd @ 25° C. ka @ 37° C. kd @ 37° C. ka @37° C. kd @ 37° C. XG2- (nM) - (nM) - (nM) - (nM) - (nM) - (nM) - xxx34mer 34mer 34mer 34mer 84mer 84mer 011 2.3 × 10⁶ 2.2 × 10⁻³ 3.6 × 10⁶1.3 × 10⁻² ND ND 026 1.9 × 10⁶ 5.8 × 10⁻³ 3.1 × 10⁶ 2.9 × 10⁻² ND ND 0451.1 × 10⁷ 5.5 × 10⁻³ 1.1 × 10⁷ 2.2 × 10⁻² ND ND 057 5.6 × 10⁶ 5.7 × 10⁻³1.1 × 10⁷ 1.1 × 10⁻² ND ND 086 2.2 × 10⁶ 1.0 × 10⁻² 4.3 × 10⁶ 7.2 × 10⁻²ND ND 124 7.9 × 10⁶ 4.3 × 10⁻³ 7.3 × 10⁶ 2.4 × 10⁻² ND ND 140 5.9 × 10⁶4.6 × 10⁻³ 5.2 × 10⁶ 2.7 × 10⁻² ND ND 163 1.5 × 10⁶ 4.5 × 10⁻³ 3.4 × 10⁶2.7 × 10⁻² 3.8 × 10⁵ 1.6 × 10⁻² 168 9.3 × 10⁵ 2.5 × 10⁻³ 3.1 × 10⁶ 8.1 ×10⁻³ 5.2 × 10⁵ 5.2 × 10⁻³ 168g2/ 3.0 × 10⁶ 8.3 × 10⁻⁴ 5.3 × 10⁶ 3.4 ×10⁻³ ND ND 183k 183 4.2 × 10⁶ 3.5 × 10⁻⁴ 1.5 × 10⁷ 1.3 × 10⁻³ 2.0 × 10⁶2.0 × 10⁻³ 214 1.5 × 10⁵ 1.2 × 10⁻⁴ 2.6 × 10⁵ 1.1 × 10⁻⁴ 1.6 × 10⁵ 2.5 ×10⁻⁴ 225 2.9 × 10⁵ 1.0 × 10⁻⁴ 8.9 × 10⁵ 5.5 × 10⁻⁴ 2.7 × 10⁵ 1.9 × 10⁻³238 1.7 × 10⁵ 1.0 × 10⁻⁴ 7.6 × 10⁵ 1.8 × 10⁻⁴ 2.1 × 10⁵ 8.2 × 10⁻⁴ 2627.4 × 10⁶ 6.8 × 10⁻³ 1.7 × 10⁷ 4.2 × 10⁻² ND ND 275 4.9 × 10⁵ 9.8 × 10⁻⁵ND ND ND ND 302 ND ND ND ND ND ND

Neutralization in vitro of PTH bioactivity with anti-PTH mAb 183. PTHits biological signal to responsive cells through the N-terminal 34amino acids. Anti-PTH mAb 183 binds to amino acids 18-34 and thereforemight be able to inhibit biological activity. As a model system, the ratosteoblastic cell line UMR-106 was used. Human and Rat PTH binds toUMR-106 cells and activates the PTH receptor. Upon activation, thereceptor increases the level of intracellular calcium. Intracellularcalcium was monitored by the change of fluorescence in cells loaded witha calcium sensitive fluorescent dye, which was measured by FLuorometricImaging Plate Reader (FLIPR). To determine whether anti-PTH mAb 183could neutralize PTH effect, the antibody was pre-incubated with PTH andits effect on PTH-induced calcium influx in UMR-106 cells was detectedby FLIPR. Anti-PTH mAb 183 blocked the calcium influx induced by 200 nMof human PTH 1-34 in a dose-dependent manner. The IC50 value foranti-PTH mAb 183 was approximately 100 nM as demonstrated in FIG. 1.

Neutralization in vivo of PTH bioactivity with anti-PTH mAb 183. Theability of anti-PTH mAb 183 to neutralize human PTH in vivo was assessedby infusing the synthetic human PTH 34mer into rats and subsequentlyadministering anti-PTH mAb 183 or PBS. The 34mer was administeredsubcutaneously via a 1-week osmotic pump (50 mcg/kg/day) starting onStudy Day 1. The hypercalcemic response to the infused PTH was used as abiomarker to assess neutralization of PTH bioactivity by the anti-PTHantibody. Anti-PTH mAb 183 (3 or 10 mg/kg) or an isotype matched controlantibody (PK 16.3.1, 10 mg/kg) was administered on Study Day 3 aftersevere hypercalcemia had developed in the rats. Anti-PTH mAb 183reversed the hypercalcemic effects of infused PTH for the entire courseof the experiment at both dose levels (FIG. 2).

Example 3 Structural Analysis of Anti-PTH Antibodies

The variable heavy chains and the variable light chains for theantibodies shown in Table 1 above were sequenced to determine their DNAsequences. The complete sequence information for all anti-PTH antibodiesare shown in the sequence listing submitted herewith, includingnucleotide and amino acid sequences for each gamma and kappa chaincombination.

The variable heavy chain nucleotide sequences were analyzed to determinethe VH family, the D-region sequence and the J-region sequence. Thesequences were then translated to determine the primary amino acidsequence and compared to the germline VH, D and J-region sequences toassess somatic hypermutations. The primary amino acid sequences of allthe anti-PTH heavy chains are shown in FIG. 3. The germline sequencesare shown above and the mutations are indicated with the new amino acidsequence. Amino acids in the sequence that were identical to theindicated germline sequence are indicated with a dash (−). The lightchain was analyzed similarly to determine the V and the J-regions and toidentify any somatic mutations from germline light chain sequences (FIG.4).

Example 4 Use of Anti-PTH Antibodies as a Diagnostic Agent

Detection of PTH Antigen in a Sample

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of PTHantigen in a sample may be developed. In the assay, wells of amicrotiter plate, such as a 96-well microtiter plate or a 384-wellmicrotiter plate, are adsorbed for several hours with a first monoclonalantibody directed against the antigen. The immobilized antibody servesas a capture antibody for any of the antigen that may be present in atest sample. The wells are rinsed and treated with a blocking agent suchas milk protein or albumin to prevent nonspecific adsorption of theanalyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample may be, for example, a serum sample from asubject suspected of having levels of circulating antigen considered tobe diagnostic of pathology.

After rinsing away the test sample or standard, the wells are treatedwith a second fully human monoclonal anti-PTH antibody that is labeledby conjugation with biotin. The labeled anti-PTH antibody serves as adetecting antibody. After rinsing away excess second antibody, the wellsare treated with avidin-conjugated horseradish peroxidase (HRP) and asuitable chromogenic substrate. The concentration of the antigen in thetest samples is determined by comparison with a standard curve developedfrom the standard samples.

This ELISA assay provides a highly specific and very sensitive assay forthe detection of the PTH antigen in a test sample.

Determination of PTH Concentration in a Patient Sample

A sandwich ELISA is developed to quantify PTH levels in human bloodserum. The two monoclonal anti-PTH antibodies used in the sandwichELISA, recognize different epitopes on the PTH molecule. The ELISA isperformed as follows: 50 μl of capture anti-PTH antibody in coatingbuffer (0.1 M NaHCO₃, pH 9.6) at a concentration of 2 μg/mL is coated onELISA plates (Fisher). After incubation at 4° C. overnight, the platesare treated with 200 μl of blocking buffer (0.5% BSA, 0.1% Tween 20,0.01% Thimerosal in PBS) for 1 hr at 25° C. The plates are washed (3×)using 0.05% Tween 20 in PBS (washing buffer, WB). Normal or patient sera(Clinomics, Bioreclaimation) are diluted in blocking buffer containing50% human serum. The plates are incubated with serum samples overnightat 4° C., washed with WB, and then incubated with 100 μl/well ofbiotinylated detection anti-PTH antibody for 1 hr at 25° C. Afterwashing, the plates are incubated with HRP-Streptavidin for 15 min,washed as before, and then treated with 100 μl/well ofo-phenylenediamine in H₂O₂ (Sigma developing solution) for colorgeneration. The reaction is stopped with 50 μl/well of H₂SO₄ (2M) andanalyzed using an ELISA plate reader at 492 nm. Concentration of PTHantigen in serum samples is calculated by comparison to dilutions ofpurified PTH antigen using a four-parameter curve-fitting program.

Example 5 Pharmacokinetic and Pharmacodynamic Study of the Effects ofAnti-PTH Antibodies on Cynomolgus Parathyroid Hormone InducedHypercalcemia in Cynomolgus Monkeys

The following study was performed to assess the effects of a singleintravenous bolus injection of mAb-183 in cynomolgus monkeys infusedwith intact cynomolgus parathyroid hormone [cynoPTH (1-84)]. In thisstudy PTH was infused continuously for 17 days to mimic PTHhypersecretion in patients with hyperparathyroidism. Cynomolgus PTH,which has comparable affinity to mAb 183 as human PTH, was used insteadof human PTH to avoid immunogenicity during the extended infusionperiod. Calcium was measured as a biomarker to assess in vivoneutralization of cynoPTH (1-84) by mAb 183.

The objectives of this study were: to demonstrate in vivo neutralizationof intact PTH in a non-human primate, as measured by suppression of thecalcemic effect of continuously infused cynoPTH (1-84); to determine theduration of action of mAb 183 in a primate; to demonstrate dose-responserelationship for mAb 183; to relate serum cynoPTH (1-84) levels, asmeasured by a commercial immunoassay, to observed effects on serumcalcium; and to evaluate the pharmacokinetics of mAb 183.

CynoPTH (1-84) was obtained by custom synthesis from C. S. Bio Company,Inc. (San Carlos, Calif.). Six male and six female monkeys were randomlyassigned by body weight to three groups (2 animals/sex/group). To inducehypercalcemia, each animal received 24 μg/day cynoPTH (1-84) starting onDay 1 via Harvard syringe pump mediated continuous intravenous infusionfor 17 days. The infusion rate used was determined in a pilot study toproduce sustained hypercalcemia with total serum calcium concentrationsin the range of 13 to 15 mg/dL. The infusion solution was replenishedtwice daily.

On Day 3, animals received a single dose of IgG2 Control PK16.3.1 (Group1), 1 mg/kg mAb 183 (Group 2), or 4 mg/kg mAb 183 (Group 3). The studydesign and treatment groups are summarized in Table 13.

TABLE 13 Dose Dose Number Level Concen- Dose of Dose (mg/ trationVolume^(b) Animals/ Group Treatment Route^(a) kg) (mg/mL) (mL/kg) Sex 1PK16.3.1 IV 4 5.20 0.77 2/male Control 2/female 2 mAb 183 IV 1 5.13 0.192/male 2/female 3 mAb 183 IV 4 5.13 0.78 2/male 2/female ^(a)Intravenousdose administered as a bolus injection. ^(b)Dose volume estimated basedon the average body weight for the 12 animals on the study.

Serum was collected on Days 1 (prior to pump implantation), 3 (prior tointravenous dose), 4, 5, 6, 7, 8, 9, 11, 13, 15, and 17 for measurementof serum total calcium (tCa), ionized calcium (iCa), PTH, and mAb 183levels. Normal calcium ranges were defined by the Day 1 baseline mean(all animals) ±3 standard deviations. Serum PTH was measured by achemiluminescent immunoassay (Nichols Advantage Intact PTH Assay,Nichols Institute Diagnostics, San Clemente, Calif.).

FIG. 5 shows the mean (SEM) serum total (FIG. 5A) and ionized calcium(FIG. 5B) profiles in the subject monkeys. The shaded area indicatesnormal calcium ranges.

The data indicate that control animals experienced stable hypercalcemiafor 17 days of infusion. Calcium was elevated by approximately 30% ormore for both tCa (˜14 mg/dL) and iCa (˜6.5 mg/dL). On Day 3 prior todosing with mAb 183 or the control article, an equivalent level ofhypercalcemia was achieved in all groups.

Monoclonal anti-PTH antibody 183 produced sustained, dose-dependentsuppression of hypercalcemia. Following a single 1 mg/kg dose of mAb183, tCa levels were decreased to ˜12.5 mg/kdL (above the normal range)for 5 days. In the 4 mg/kg group, tCa was decreased to the upper end ofthe normal range for 8 days. Two weeks after dosing, tCa (˜13 mg/dL) wasstill below the level of control (˜14.5 mg/dL). Similar trends wereobserved for the iCa levels. (See FIGS. 5A and 5B)

The measurement of serum PTH levels from the study are presented in FIG.6. Serum PTH increased from approximately 60 pg/mL at baseline toapproximately 250 pg/mL in control treated animals. On Day 3, PTH levelsin all dose groups were lower than baseline (˜50 pg/mL) which wasattributed to the infusion syringe having run out of solution just priorto blood collection. The decreased PTH levels relative to baselineprobably reflect a homeostatic decrease in endogenous PTH secretion,consistent with the Day 3 hypercalcemia. A dose of 1 mg/kg mAb 183reduced PTH levels to about 150 pg/mL for 3-4 days following dosing,compared to 250 pg/mL in control animals. The slight reduction in PTHover this period was in good agreement with modest calcium suppressionobserved over 5 days following dosing. Animals treated with 4 mg/kg mAb183 had reductions in PTH levels to around 75-100 pg/mL for 8 days afterdosing, which corresponded to suppression of calcium to the upper end ofthe normal range for 8 days. In the 4 mg/kg group, PTH was still belowthe level of control 2 weeks after dosing, consistent with a reductionin serum calcium relative to control. In both mAb 183 treatment groups,the kinetics of calcium suppression corresponded closely to the kineticsof PTH suppression.

In summary, mAb 183 produced dose-dependent and prolonged suppression ofinfused cynoPTH (1-84). The study confirms that mAb 183 neutralizes thebioactivity of intact PTH in primates, and confirms the findings seen inrats infused with the huPTH (1-34) N-terminal fragment. Additionally,the excellent agreement between the measured serum PTH levels and thechanges in serum calcium after dosing indicates that the commercialimmunoassay used in this study can successfully measure levels of free,bioactive PTH in vivo in the presence of circulating mAb 183.

Example 6 Synergistic Activity of mAb sc275 in Combination with mAb 183in Healthy Cynomolgus Monkeys

Five, naïve-to-antibodies, female cynomolgus monkeys were assigned foracclimation. The animals were screened for health status and underwenthematology and serum chemistry and parathyroid hormone (PTH) screening.Four selected animals were then assigned to treatment groups as noted inTable 14.

Two female cynomolgus monkey were assigned to each treatment group for atotal study length of 28 days. Animals were dosed according to thescheme shown in Table 14.

TABLE 14 Dose Dose Group/ Level Route Dose Conc. Number of Color (mg/ ofVolume (mg/ Female Code Test Article kg) Admin. (mL/kg)^(a) mL) Monkeys1/white ABX10241 5 IV 0.5 & 10 & 2 SC275 5 4.95 1.01 2/yellow ABX1024110 IV 1.0 10 2 ^(a)Dose volume (mL/kg) will be based on the most recentbody weight and rounded to the nearest syringe increment according toSNBL USA SOP.

FIG. 7 shows the serum iCa as a percent change from the baseline foureach of the four dosages over the course of the investigation. As shown,the use of the two classes of antibodies together resulted in a loweringof serum iCa that was more profound and longer lasting than that inducedby the use of mAb 183 alone.

Example 7 Epitope Mapping with Alanine Scanning

A custom-made peptide array was obtained from Sigma-Genosys. A series of23, 12-mer peptides were synthesized with peptides spanning residues1-34 of the PTH sequence. Each consecutive peptide was offset by oneamino acid from the previous one yielding a nested, overlapping library.The membrane carrying the 23 peptides was probed with anti-PTH antibody183 (1 mg/ml) and detected with HRP-conjugated secondary anti-human IgGantibody using enhanced chemiluminescence (ECL).

To further refine the contact residues involved in the binding ofanti-PTH mAb 183 to PTH, an alanine-scanning mutation approach wasemployed using a custom-peptide array. The array contained the peptidesin Table 15, and bound to mAb 183 with the stated results.

TABLE 15 Binding of mAb 183 to Amino Acids 20-28 of PTH PEPTIDE BINDINGRESULT SEQ ID NO: RVEWLRKKL Binding to the (SEQ ID NO: 99) wild-typepeptide was observed as expected. AVEWLRKKL No binding (SEQ ID NO: 102)RAEWLRKKL No binding (SEQ ID NO: 103) RVAWLRKKL No binding (SEQ ID NO:104) RVEALRKKL Binding (SEQ ID NO: 105) RVEWARKKL No binding (SEQ ID NO:106) RVEWLAKKL No binding (SEQ ID NO: 107) RVEWLRAKL Binding (SEQ ID NO:108) RVEWLRKAL No binding (SEQ ID NO: 109) RVEWLRKKA No binding (SEQ IDNO: 110)

Conclusions: The amino acids RVE_LR_KL appear to be required for theability of anti-PTH mAb 183 to bind to PTH. The important amino acidsfor antibody binding corresponded to amino acids 20, 21, 22, 24, 25, 27and 28.

Example 8 Treatment of Parathyroid Carcinoma in a Human Patient byAdministering Anti-PTH Antibodies

Parathyroid carcinoma is a very rare tumor with approximately 200 casesreported in the literature. Tumors producing PTH are highlydifferentiated and slow growing. The clinical course of the disease canextend over years. Symptoms and mortality reflect the metabolicconsequences of hyperparathyroidism rather than tumor burden. Surgicalexcision is the treatment of choice in this population. Medicalmanagement of metastatic disease or inoperable tumors includesbisphosphonates, calcitonin, chemotherapeutic drugs targetingosteoclasts and, most recently, cinacalcet, a calcimimetic.

mAb 183 is a fully human, high-affinity, monoclonal antibody directedagainst the 1-34 amino acid region of parathyroid hormone (PTH) thatneutralizes the activity of PTH in vitro and in vivo. mAb 183 was usedto treat a 38 year-old male with a 14-year history of inoperableparathyroid carcinoma. The patient had been treated with calcitonin(severe allergic reaction), bisphosphonates (pamidronate and zoledronicacid) and cinacalcet without control of his disease.

Hemodialysis was initiated to treat progressive renal insufficiency.Following atraumatic, bilateral femoral neck fractures, treatment withmAb 183 was initiated under an emergency use investigational new drug(“IND”) mAb 183 was administered by intravenous bolus at increasingdoses over a two-week period to reduce pre-dose plasma unbound PTH to<200 pg/ml. mAb 183 was administered in doses of 25 mg (Day 1), 50 mg(Day 4), 100 mg (Day 8), and 200 mg (Day 11, Day 22, Day 32, andapproximately weekly thereafter). Plasma unbound PTH (measured using theNichols Intact Assay), collagen N-telopeptides (NTx), and 1,25 (OH)2vitamin D (vit D) and pharmacokinetic samples were obtained before eachdose, 24 hours post-dose and approximately 24 hours prior to the nextdose during the dose-escalation phase. Serum iCa was obtained every 8hours during the dose-escalation phase.

The patient continued to receive 3-4 times weekly hemodialysis andintermittent doses of zoledronic acid. Treatment with mAb 183 resultedin a profound, dose- and exposure-dependent reduction in plasma unboundPTH from a pre-dose level of ˜1400 pg/ml to the targeted range at a doseof 200 mg per week (as shown in FIG. 8). Plasma NTx levels were reducedfollowing each dose of mAb 183 suggesting bone turnover was impacted bythe treatment. In addition, vitamin D levels were reduced by thetreatment. The dose of mAb 183 was further escalated to 500 mg per weekresulting in a clinically significant reduction in serum calcium (>3mg/dL) to within the normal range allowing resumption of hemodialysisagainst a normal calcium bath (levels of serum calcium are shown in FIG.9). The reduction in serum calcium was accompanied by reductions in 1,25 dihydroxy vitamin D and plasma N-telopeptides (shown in FIGS. 10 and11 respectively). No drug related toxicities were observed during orafter the administration of mAb 183 and the patient reported improvementof his bone pain. It was observed that mAb 183 administered byintravenous bolus at doses ranging from 25-500 mg per week over 6 monthswas well tolerated. No human anti-human antibodies were detected. Thefindings support the potential benefit of mAb 183 in the treatment ofhyperparathyroidism secondary to parathyroid carcinoma.

Example 9 Treatment of Parathyroid Hormone Secondary Hyperparathyroidism(SHPT) Patients

Secondary hyperparathyroidism (SHPT), a common condition in patientswith end-stage renal disease (ESRD) on hemodialysis, is associated withrenal osteodystrophy and can lead to significant morbidity. Currentapproaches to SHPT management include reducing hyperphosphatemia,maintaining normal serum calcium concentrations, and replacing1,25(OH)₂D₃. Therapeutic interventions designed to achieve these goalsinclude vitamin D analogs, calcium supplementation, calcimimetics andnonabsorbable phosphate binders. While current therapies offer importantbenefits, they often have limited efficacy, poor tolerability and cancontribute to significant toxicities, such as hypercalcemia and ectopiccalcification. Additionally, the practical application of sometreatments may be limited by drug-drug interactions or poor compliance.

mAb 183 is a high affinity monoclonal antibody directed against theregion of human parathyroid hormone (PTH) that included amino acids1-34. mAb 183 is specific for human and non-human primate PTH. Inprimate and rodent models of hyperparathyroidism induced by continuousadministration of PTH, mAb 183 reversed the metabolic consequences ofhyperparathyroidism in a dose-dependent manner. In the primate model, asingle 4 mg/kg dose of mAb 183 produced a sustained reduction in plasmaunbound PTH (non-antibody complexed PTH measured with the Nichols IntactAssay) and normalized serum Ca for up to two weeks relative to controlanimals.

On the basis of these results, a randomized, double-blind, single-dose,dose escalation study in hemodialysis patients with SHPT was initiated.Eligibility criteria include plasma PTH >300 pg/mL (Nichols IntactAssay), serum corrected Ca>9 mg/dL, and a Kt/V>1.2. Vitamin D or vitaminD analogs, oral phosphate binders, and cinacalcet were permittedprovided the doses were stable for at least 2 weeks prior to treatment.Planned dose cohorts include 30, 100, 200, and 300 mg of mAb 183; thestarting dose of 30 mg was less than the no-effect level in preclinicalstudies. Within each cohort subjects were randomized 3:1 to mAb 183 orplacebo. mAb 183 was administered by IV push after hemodialysis andpatients were monitored in a Phase 1 unit for 3 days post treatment.

Four patients received 30 mg and 8 patients received 100 mg of mAb 183.The subjects were monitored in a Phase 1 unit for 72 hours after dosingbetween hemodialysis sessions. The subjects were then followed for 16weeks after discharge from the Phase 1 unit. The patients' serumchemistries were measured approximately every 8 hours during thein-house observation period. Unbound iPTH was measured at 1 hour, 24hours and 72 hours after dosing and weekly after discharge from thePhase 1 unit.

It was observed that single 30 and 100 mg doses of mAb 183 administeredby intravenous bolus were safe and well tolerated. Treatment with mAb183 resulted in: dose dependent suppression of unbound iPTH as shown inFIG. 12; dose dependent reduction in serum calcium as shown in FIGS. 13,14, and 15 (FIG. 13 shows the actual ionized calcium levels whereasFIGS. 14 and 15 show corrected calcium levels); and reduction in thecalcium—phosphorus product. It was further observed that a single doseof mAb 183 was effective in reducing unbound iPTH to the Kidney DiseaseOutcome Quality Initiative (K/DOQI) range across a broad range ofdisease severity (as shown in FIG. 16). A single 100 mg dose of mAb 183suppressed unbound iPTH below 300 pg/mL for 7 days in 88% of subjects(as shown in FIG. 17). Additionally, a single 100 mg dose of mAb 183reduced serum bAP in patients with elevated bAP at baseline (as shown inFIG. 18). The pharmacokinetics of unbound mAb 183 were found to belinear with dose (as shown in FIG. 19). No human anti-human antibodieswere detected in any subject at any time point.

Example 10 Treatment of Secondary Hyperparathyroidism with AntibodyCombination

To determine the in vivo effects of anti-PTH antibody treatment in humanpatients with secondary hyperparathyroidism, a patient with end-stagerenal disease is selected for treatment. The selected patient is ondialysis for at least four months prior to treatment. The patient has ahistory of elevated serum PTH values of greater than 400 pg/mL when notreceiving therapy.

The patient is injected over several weeks with an effective amount of acombination of mAb 183 and sc 275 in a pharmaceutical preparation,preferably at 10 mg/kg of each antibody. At periodic times during thetreatment, the patient is monitored to determine levels of serumcalcium, phosphorus, osteocalcin, bone alkaline phosphatase, unboundactive PTH, total PTH (antibody bound and unbound), and radial, hip andspinal bone mineral density.

Analysis of the clinical data shows that the combination treatmentsignificantly reduces the level of circulating active PTH as determinedin the above example. The treated patient also shows a significantdecrease in the serum levels of osteocalcin and bone alkalinephosphatase. The treated patient may demonstrate a transient reductionin serum calcium following the first dose or with dose escalation. Overtime, radial, hip and spinal bone mineral density will increase comparedto baseline.

CONCLUSION

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The inventiondescribed herein is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain embodiments of the invention and any constructsthat are functionally equivalent are within the scope of this invention.The deposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any embodiment of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

1. A method of reducing elevated circulating levels of parathyroidhormone (PTH) in a patient, comprising: identifying a patient in need oftreatment for elevated circulating levels of PTH; and administering tosaid patient a therapeutically effective dose of a fully humanmonoclonal antibody, or binding fragment thereof, that binds with anaffinity to PTH of less than 100 pM, and reduces the circulating levelsof PTH below 300 pG/ml for at least about seven days, wherein said fullyhuman monoclonal antibody is mAb
 183. 2. The method of claim 1, whereinsaid elevated circulating levels of PTH are associated withhyperparathyroidism.
 3. The method of claim 2, wherein saidhyperparathyroidism is primary hyperparathyroidism.
 4. The method ofclaim 2, wherein said hyperparathyroidism is secondaryhyperparathyroidism.
 5. The method of claim 1, further comprisingtreating said patient with a second fully human monoclonal antibody orbinding fragment thereof that binds to PTH.
 6. The method of claim 5,wherein said second fully human monoclonal antibody or binding fragmentthereof binds amino acids 1-7 of PTH.
 7. A method of effectivelytreating hypercalcemia in a patient, comprising: identifying a patientin need of treatment for hypercalcemia; administering to said patient atherapeutically effective dose of a first fully human monoclonalantibody or binding fragment thereof that specifically binds toparathyroid hormone (PTH), wherein the administration of said monoclonalantibody or binding fragment thereof reduces serum calcium levels below12 mg/dL, and wherein said patient is a human; and treating said patientwith a second fully human monoclonal antibody or binding fragmentthereof that binds to PTH, wherein said second fully human monoclonalantibody is mAb sc275.
 8. A method of effectively treating hypercalcemiain a patient, comprising: identifying a patient in need of treatment forhypercalcemia; and administering to said patient a therapeuticallyeffective dose of a fully human monoclonal antibody or binding fragmentthereof that specifically binds to parathyroid hormone (PTH), whereinthe administration of said monoclonal antibody or binding fragmentthereof reduces serum calcium levels below 12 mg/dL, and wherein saidpatient is a human, wherein said fully human monoclonal antibody is mAb183.
 9. A method of effectively treating parathyroid carcinoma in apatient, comprising: identifying a patient in need of treatment forparathyroid carcinoma; administering to said patient a therapeuticallyeffective dose of a first fully human monoclonal antibody or bindingfragment thereof that specifically binds to parathyroid hormone (PTH);wherein the administration of said antibody or binding fragment thereofresults in the sustained reduction of circulating PTH below 300 pg/mlfor a period of at least three days, and wherein said patient is ahuman; and treating said patient with a second fully human monoclonalantibody or binding fragment thereof that binds to PTH, wherein saidsecond fully human monoclonal antibody is mAb sc275.
 10. A method ofeffectively treating parathyroid carcinoma in a patient, comprising:identifying a patient in need of treatment for parathyroid carcinoma;and administering to said patient a therapeutically effective dose of afully human monoclonal antibody or binding fragment thereof thatspecifically binds to parathyroid hormone (PTH); wherein theadministration of said antibody or binding fragment thereof results inthe sustained reduction of circulating PTH below 300 pg/ml for a periodof at least three days, and wherein said patient is a human, whereinsaid fully human monoclonal antibody is mAb
 183. 11. The method of claim1 wherein said binding fragment is selected from the group consisting ofFab, Fab′, F(ab′)₂, and F_(v).
 12. The method of claim 8, wherein saidbinding fragment is selected from the group consisting of Fab, Fab′,F(ab′)₂, and F_(v).
 13. The method of claim 10, wherein said bindingfragment is selected from the group consisting of Fab, Fab′, F(ab′)₂,and F_(v).
 14. The method of claim 1, wherein said PTH levels arereduced below 200 pg/ml.
 15. The method of claim 9, wherein the PTH isreduced for at least seven days.
 16. The method of claim 9, wherein thePTH is reduced for at least eleven days.
 17. The method of claim 1,wherein the PTH is reduced for at least eleven days.
 18. The method ofclaim 10, wherein the PTH is reduced for at least seven days.
 19. Themethod of claim 10, wherein the PTH is reduced for at least eleven days.20. The method of claim 7, wherein said binding fragment of said firstfully human monoclonal antibody is selected from the group consisting ofFab, Fab′, F(ab′)₂, and F_(v).
 21. The method of claim 7, wherein saidbinding fragment of said second fully human monoclonal antibody isselected from the group consisting of Fab, Fab′, F(ab′)₂, and F_(v). 22.The method of claim 7, wherein said first fully human monoclonalantibody is mAb183.
 23. The method of claim 9, wherein said bindingfragment of said first fully human monoclonal antibody is selected fromthe group consisting of Fab, Fab′, F(ab′)₂, and F_(v).
 24. The method ofclaim 9, wherein said binding fragment of said second fully humanmonoclonal antibody is selected from the group consisting of Fab, Fab′,F(ab′)₂, and F_(v).
 25. The method of claim 9, wherein said first fullyhuman monoclonal antibody is mAb183.
 26. The method of claim 5, whereinsaid second fully human monoclonal antibody is mAb sc275.
 27. The methodof claim 5, wherein said binding fragment of said second fully humanmonoclonal antibody is selected from the group consisting of Fab, Fab′,F(ab′)₂, and F_(v).